Variation in Tropical Tree Seedling Survival, Growth

Home , Myristicaceae, Virola surinamensis

Variation in Tropical Tree Seedling Survival, Growth, and Colonization by Arbuscular Mycorrhizal Fungi near Conspecific Adults: Field and Shadehouse Experiments in Panama

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Jenalle Laree Eck, B.S.

Graduate Program in Evolution, Ecology and Organismal Biology

The Ohio State University

2017

Dissertation Committee:

Allison A. Snow, Advisor

Liza S. Comita, Co-advisor

P. Enrico Bonello

Simon A. Queenborough

Copyrighted by

Jenalle Laree Eck

2017

Abstract

The Janzen-Connell hypothesis highlights the important role of species-specific natural enemies like pathogens and herbivores in maintaining species diversity within plant communities by limiting the survival and growth of seedlings near conspecific adult plants. The extent to which natural enemies reduce conspecific seedling performance is thought to influence species richness and relative abundance within plant communities, but within-species variation in this process could also affect plant diversity at the species or population level. Variation in the strength of natural enemy effects among conspecific seedlings could occur due to factors such as the degree of relatedness between conspecific seedlings and adults (i.e., their level of shared susceptibility), or due to the characteristics of the adult that determine natural enemy accumulation (e.g., reproduction), but the causes of such variation and its consequences for patterns of plant diversity have seldom been explored.

I conducted experiments with tropical tree species on Barro Colorado Island,

Panama, to test new hypotheses about the causes of variation in survival and growth of conspecific seedlings near adult plants, and to examine how this variation could structure patterns of plant diversity. In Chapter 1, I conducted a shadehouse experiment with the tropical tree species Virola surinamensis to test the hypothesis that highly specialized soil microbial communities accumulate around adults within species and reduce the

ii performance of offspring seedlings relative to non-offspring conspecifics. In Chapter 2, I conducted a field experiment with four tropical tree species to test the hypothesis that seedling performance is reduced beneath the canopies of their own parent trees than beneath those of different conspecific adults. In Chapter 3, I conducted shadehouse and field experiments with V. surinamensis to test the hypothesis that seed production reduces the performance of conspecific seedlings near female trees relative to males by increasing the density of natural enemies beneath females.

I found evidence that the soil microbial communities beneath female trees of V. surinamensis reduce the growth and colonization by mutualistic arbuscular mycorrhizal fungi (AMF) of their own offspring seedlings relative to non-offspring conspecifics. This suggests that the genetic relationship between conspecific seedlings and adults determines the outcome of seedling interactions with soil microbes. I did not find evidence that this relationship determined the survival or growth of seedlings of four species beneath conspecific adults in the field. Thus, while highly specialized interactions between soil microbes and tropical trees can occur, when seedlings are exposed to all potential natural enemies and variable environmental conditions, such effects may not be the most important determinants of seedling survival or growth. Lastly, I did not find evidence that seed production by female trees reduces conspecific seedling survival, growth, or colonization by AMF near females relative to males, suggesting that seed production does not alter natural enemy densities or interactions with seedlings in a meaningful way. These findings improve our understanding of the Janzen-Connell hypothesis by providing evidence that interactions between tropical trees and soil

iii microbes can be highly specialized and could help to maintain genetic diversity within tropical tree populations.

People, plants, animals, places, moments, traces, you lit a way.

Acknowledgments

I am humbled to acknowledge my gratitude to a great many people that lit a way here. You have created an astonishing experience of time, place, and life.

This journey began with family and place. My mother, Evelyn Dee Evans-Eck, and father, James Edward Eck, Jr., have been wonderfully multifaceted role models of love and hard work, and an endless source of strength, support, and fun. My loving grandparents, the late Richard Edwin Evans, Sr., of Ellensboro, WV and Shirley Jean

Evans (York) of Cheboygan, MI, and James Edward Eck, Sr. and Lydia Helen Eck

(Bahls) of Toledo, OH, each cared for me and taught me in their own best way. My dear aunts, Marcia Eck and Liz Morrow (Evans), have imparted their unique perspectives and enjoyments of life. The countless hours I spent learning from and being cared for by each of these people, at home and in the outdoors, allowed me to grow in a great many areas. I am eternally grateful to each of these people for their roles in nurturing my development, and for their tolerance and encouragement of a life spent pursuing dreams, even when those dreams have taken me far from home.

My advisor, Liza Comita, opened the door for me to conduct graduate research in the tropical forests of Panama six years ago, and since then, has done everything in her power to impart on me the skills and qualities needed to become a scientist. She has been generous with her time and knowledge, patient and constructive in her approach to

vi advising, understanding of the trials of graduate school, and an all-around excellent professional role model. Thank you, Liza, for opening the doors to Ohio State, Panama, the Smithsonian Tropical Research Institute, Yale, Penn State, and beyond, and equipping me to step through them confidently; it has been an honor to be your student.

My passion for research in plant ecology has been cultivated by inspiration and opportunity granted to me by several other skilled, dedicated mentors and teachers.

Allison Snow, and her lab manager, Patty Sweeney, were the first to introduce me to research in plant ecology during my undergraduate degree at Ohio State. In the Snow lab,

I learned to apply my love of plants and the outdoors to research in the field, lab, and greenhouse, and I became interested in a career in plant ecology. I was granted another opportunity to develop as a plant scientist by Anne Dorrance, who hired me to study soybean disease resistance in her lab in Wooster, OH. The skills and perspectives I gained while in the Dorrance lab further shaped the direction of my own research. As a graduate student taking courses at Ohio State, I benefitted greatly from the instruction of

P. Enrico Bonello, Simon Queenborough, Libby Marschall, Maria Miriti, Peter Curtis,

Lisle Gibbs, Charles Goebel, Kristin Mercer, Terry Niblack, and Dan Herms. The members of my candidacy and dissertation committee, Enrico Bonello, Simon

Queenborough, and Allison Snow, in addition to Liza Comita, have provided helpful direction and feedback on my research goals and proposals and manuscript drafts. At the

Smithsonian Tropical Research Institute in Panama, I was fortunate to receive research support and mentorship from Scott Mangan, E. Allen Herre, S. Joseph Wright, Helene

Muller-Landau, and Egbert Giles Leigh, Jr. At Penn State University, I am grateful to be

vii embarking on a new genetics project with the expertise, efforts, and support of James

Marden, Claude dePamphilis, and Howard Fescemyer. My deepest gratitude is extended to each of these mentors for their role in my professional development.

My professional peers, the graduate students and post-docs I have been lucky to meet and learn with along the way, have been an inspiration, as well as a source of help, support, and comradery. Special thanks are extended to the past and present members of the Comita lab, who have been my academic family at Ohio State and Yale: Stephen

Murphy, Meghna Krishnadas, Juan Carlos Penagos Zuluaga, Kara Salpeter, Noelle

Beckman, Andrew Muehliesen, Dan Johnson, Livia Audino-Dorneles, Yan Zhu, Megan

Sullivan, Anna Sugiyama, Harikrishnan Venugopalan Nair Radhamoni, and Madelon

Case. The many talented, dedicated students I met at the Smithsonian Tropical Research

Institute in Panama have forever shaped my life in the years that I spent working, living, and learning alongside of them on Barro Colorado Island and in Gamboa: Dana

Frederick, Carolyn Delevich, Kristen Becklund, Matthew Lutz, Katie Heineman, KC

Cushman, Lourdes Hernández, Maria Muriel Garcia, Jerry Schneider, Krystaal McClain,

Peter Marting, Victoria Weaver, Merlin Sheldrake, Luis Beltran, Tom Bochynek, Chris

Reid, Justin Shaffer, Emily Francis, Natalie Christian, Callum Kingwell, Julian Schmid,

Georg Eibner, Eli Rodriguez, Eric Griffin, Camilo Zalamea, Carolina Sarmiento, Laura

Walker, Jacquelline Dillard, Meghan Strong, Max Adams, Adriana Corrales Osario, Sara

Fern Leitman Neihaus, Margarita Cecilia Prada, Megan Pendred, Tim Alvey, Erin Welsh,

Dan Revillini, Emily Borodkin, Ernesto Bonadies, and countless others, thank you for making my time in Panama truly cherished. Of these people, Matthew Lutz deserves

viii special mention for his years of unwavering love, support, and companionship, in

Panama and beyond. Many other graduate students and post-docs at Ohio State, Yale,

STRI and Penn State also improved my graduate experience – thank you all.

The projects described in these chapters would not have been possible without the logistical support, expertise, hard work and donated days and hours of many. A few were truly heroes. Lourdes Hernández single-handedly ensured the success of the field experiments. Camille Delavaux was instrumental in generating the arbuscular mycorrhizal fungi data. Carolyn Delevich helped me out of more than one pinch. Dara

Wilson and Blexein Contreras tended the shadehouse experiments while I was on campus at Yale. Oris Acevedo, Belkys Jimenez, and Melissa Cano provided logistical support on

BCI. Megan Sullivan and Lisa Miller helped to set up a pilot project leading to these projects. Juan Carlos Penagos Zuluaga used his skill with a sling-shot to collect leaf samples for the new genetics study.

I owe my gratitude to several institutions for providing a home for or funding for my academic training and research: the Department of Evolution, Ecology, and

Organismal Biology, Graduate School, and Council of Graduate Students at the Ohio

State University, the Smithsonian Institution and Smithsonian Tropical Research

Institute, Yale University School of Forestry and Environmental Studies, Pennsylvania

State University Department of Biology, the National Science Foundation, and the

Ecological Society of America.

Lastly, I am humbled to acknowledge the role of the forest on Barro Colorado

Island in inspiring and enabling this work. Many months spent coming to know the plants

ix and animals in this forest, and the seeds that were given to me by the trees, have been a privilege that I, here, hope to share and put to greater use.

Vita

2006...... Springfield High School, Holland, Ohio

2008 - 2010 ...... Research Aide, Dept. of Evolution, Ecology

and Organismal Biology, The Ohio State

University

2010...... B.S. Biology, The Ohio State University

2011...... Research Aide, Dept. of Plant Pathology,

The Ohio State University

2011 - 2013 ...... Graduate Teaching Associate, Dept. of

Evolution, Ecology and Organismal

Biology, The Ohio State University

2012...... Intern, Smithsonian Tropical Research

Institute

2014 – 2017...... Visiting Associate in Research, School of

Forestry and Environmental Studies, Yale

University

2015 – 2016...... Predoctoral Fellow, Smithsonian Tropical

Research Institute

2017...... Visiting Scholar, Department of Biology,

Pennsylvania State University

Publications

Murphy, S. J., Audino, L. D., Whitacre, J., Eck, J. L., Wenzel, J. W., Queenborough, S.,

& Comita, L. S. (2015) Species associations structured by environment and land-use history promote beta-diversity in a temperate forest. Ecology 96, 705-715.

Comita, L. S., Queenborough, S. A., Murphy, S. J., Eck, J. L., Xu, K., Krishnadas, M.,

Beckman, N. G., & Zhu, Y. (2014). Testing predictions of the Janzen-Connell hypothesis: a meta-analysis of experimental evidence for distance- and density-dependent seed and seedling survival. Journal of Ecology 102, 845-856.

Fields of Study

Major Field: Evolution, Ecology and Organismal Biology

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Table of Contents

Abstract ...... ii

Acknowledgments...... vi

Vita ...... xi

List of Figures ...... xv

List of Tables ...... xviii

Synthesis: Overview of Research Questions & Summary of Results...... 1

Chapter 1: Soil Microbes reduce Growth of Offspring vs. Non-offspring Conspecific

Seedlings: Experimental Evidence of a Janzen-Connell Effect within a Tropical Tree

Population ...... 11

Chapter 2: Tropical Tree Seedling Survival and Growth Near Mother and Other

Conspecific Adults in the Field...... 51

Chapter 3: Conspecific Seedling Survival, Growth, and Colonization by Arbuscular

Mycorrhizal Fungi near Female and Male Adults in a Dioecious Tropical Tree Species 85

References ...... 141

Appendix A: AM Fungi (Chapter 1) ...... 151

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Appendix B: Soil Nutrients (Chapter 1) ...... 155

Appendix C: Conspecific Seedling Density (Chapter 3) ...... 156

Appendix D: Clipping (Chapter 3) ...... 158

Appendix E: Offspring Seedlings (Chapter 3) ...... 160

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List of Figures

Figure 1: Conceptual diagram of how genotype-specific soil pathogens could promote genetic diversity in plant populations. …………………………………………………. 14

Figure 2: A spatial map of the location of the 11 maternal trees in the ‘mother vs. other’ shadehouse experiment on BCI. ……………………………………………………….. 21

Figure 3: A photograph of the ‘mother vs. other’ shadehouse experiment. …………… 24

Figure 4: A micrograph of AM fungi structures in the root of an experimental seedling. …………………………………………………………………………………………... 26

Figure 5: Biomass of offspring vs. non-offspring seedlings in conspecific soil microbial communities in the ‘mother vs. other’ shadehouse experiment. ……………………….. 33

Figure 6: Biomass of seedlings across 11 maternal seed sources and 11 soil microbial communities in the ‘mother vs. other’ shadehouse experiment. ……………………….. 34

Figure 7: Seedling growth as a function of AMF colonization in conspecific soil microbial communities in the ‘mother vs. other’ shadehouse experiment. ……………. 36

Figure 8: Plant-soil feedback among mother trees in the ‘mother vs. other’ shadehouse experiment. ……………………………………………………………………………... 39

Figure 9: Plant-soil feedback of conspecific seedlings. ……………………………….. 40

Figure 10: Colonization by AM fungi in offspring vs. non-offspring seedlings in conspecific soil microbial communities in the ‘mother vs. other’ shadehouse experiment. …………………………………………………………………………………………... 43

Figure 11: Colonization by AM fungi of seedlings across 11 maternal seed sources and 11 soil microbial communities in the ‘mother vs. other’ shadehouse experiment. ………… 44

Figure 12: A photograph of a tagged experimental seedling in a field plot. …………… 63

Figure 13: Seedling survival in mother vs. other conspecific environments in the field. …………………………………………………………………………………………... 69

Figure 14: Survival of seedlings from 19 maternal seed sources in mother and other conspecific environments the field. ……………………………………………………... 70

Figure 15: Survival of offspring vs. non-offspring conspecific seedlings near 22 focal adults in the field. ……………………………………………………………………….. 71

Figure 16: Seedling survival across conspecific seedling densities in the ‘mother vs. other’ field experiment. ………………………………………………………………………... 72

Figure 17: Seedling biomass in mother vs. other conspecific environments in the field. ..73

Figure 18: Biomass of seedlings from 19 maternal seed sources in mother and other conspecific environments the field. ……………………………………………………... 76

Figure 19: Biomass of offspring vs. non-offspring conspecific seedlings near 22 focal adults in the field. ……………………………………………………………………….. 77

Figure 20: Seedling biomass across conspecific seedling densities in the ‘mother vs. other’ field experiment. ………………………………………………………………… 78

Figure 21: A spatial map of the location of the 11 female and 4 male trees in the ‘male vs. female’ shadehouse experiment on BCI. ………………………………………………... 95

Figure 22: A photograph of the ‘male vs. female’ shadehouse experiment. …………… 99

Figure 23: A micrograph of AM fungi structures in the root of a seedling in the ‘male vs. female’ shadehouse experiment. ………………………………………………………..101

Figure 24: A spatial map of the location of the 6 female and 4 male trees in the male vs. female field experiment on BCI. ………………………………………………………. 104

Figure 25: Censusing seedling survival and biomass in male vs. female field plots. …..108

Figure 26: Biomass of seedlings in male vs. female soil microbial communities in the shadehouse. ……………………………………………………………………………. 115

Figure 27: Biomass of seedlings across 11 maternal seed sources in male vs. female soil microbial communities in the shadehouse. ……………………………………………. 116

Figure 28: Seedling biomass in the soil microbial communities of females that varied in seed production. ……………………………………………………………………….. 118

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Figure 29: Colonization by AM fungi of seedlings in male vs. female soil microbial communities. ………………………………………………………………………….. 121

Figure 30: Colonization by AM fungi of seedlings across 11 maternal seed sources in male vs. female soil microbial communities. ……………………………………………….. 122

Figure 31: Seedling colonization by AM fungi in the soil microbial communities of females that varied in seed production. ……………………………………………….. 124

Figure 32: Seedling survival near male vs. female conspecifics in the field. …………. 127

Figure 33: Survival of seedlings across 6 maternal seed sources in male vs. female environments in the field. ……………………………………………………………… 128

Figure 34: Seedling survival near male vs. female conspecifics in the field depending on conspecific seedling density. ………………………………………………………….. 129

Figure 35: Seedling biomass near male vs. female conspecifics in the field. ………… 132

Figure 36: Biomass of seedlings across 6 maternal seed sources in male vs. female environments in the field. ……………………………………………………………… 133

Figure 37: Seedling biomass near male vs. female adults in the field across conspecific seedling densities. ……………………………………………………………………... 134

Figure 38: PCA of nutrients in the soil inocula. ……………………………………... 155

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List of Tables

Table 1: Sample sizes for each combination of maternal seed source and soil microbial source in the ‘mother vs. other’ shadehouse experiment. ……………………………… 23

Table 2: Mixed-model ANCOVA of biomass in offspring vs. non-offspring seedlings in the ‘mother vs. other’ shadehouse experiment. ………………………………………... 32

Table 3: Mixed-model ANCOVA of the impact of colonization by AM fungi on seedling biomass in the ‘mother vs. other’ shadehouse experiment. ……………………………. 35

Table 4: Seedling biomass as a function of soil nutrients in the ‘mother vs. other’ shadehouse experiment. ………………………………………………………………... 37

Table 5: Average plant-soil feedback among mother trees in the ‘mother vs. other’ shadehouse experiment. ………………………………………………………………... 38

Table 6: Mixed-model ANCOVA of system-wide plant-soil feedback in the ‘mother vs. other’ shadehouse experiment. …………………………………………………………. 39

Table 7: Mixed-model ANCOVA of colonization by AM fungi in offspring vs. non- offspring seedlings in the ‘mother vs. other’ shadehouse experiment. ………………… 42

Table 8: Colonization by AM fungi as a function of soil nutrients in the ‘mother vs. other’ shadehouse experiment. …………………………………………………………. 45

Table 9: Sample sizes for each species and treatment in the ‘mother vs. other’ field experiment. ……………………………………………………………………………... 59

Table 10: Survival of seedlings in the ‘mother vs. other’ field experiment. ……………67

Table 11: Mixed-model ANCOVA of seedling survival in mother vs. other conspecific environments in the field. ……………………………………………………………… 68

Table 12: Mixed-model ANCOVA of seedling biomass in mother vs. other conspecific environments in the field. ……………………………………………………………… 74

xviii

Table 13: Sample sizes for each treatment group and each combination of maternal seed source and soil microbial inoculum source in the ‘male vs. female’ shadehouse experiment. ……………………………………………………………………………... 97

Table 14: Sample sizes for each treatment group and each combination of maternal seed source and focal adult environment in the ‘male vs. female’ field experiment. ……… 106

Table 15: Mixed-model ANCOVA of biomass of seedlings in male and female soil microbial communities in the shadehouse. …………………………………………… 115

Table 16: Mixed-model ANCOVA of the effect of seed production on seedling biomass in female soil microbial communities in the shadehouse. ……………………………. 117

Table 17: Mixed-model ANCOVA of colonization by AM fungi of seedlings in male and female soil microbial communities in the shadehouse. ………………………………. 120

Table 18: Mixed-model ANCOVA of the effect of seed production on seedling colonization by AM fungi in female soil microbial communities in the shadehouse. ... 123

Table 19: Mixed-model ANCOVA of survival of seedlings in male and female environments in the field. …………………………………………………………….. 126

Table 20: Mixed-model ANCOVA of the biomass of seedlings in male and female environments in the field. …………………………………………………………….. 131

Table 21: Generalized linear mixed-effects model summary of the effect of observer on proportion colonization by AM fungi. ……………………………………………….. 152

Table 22: Coefficient estimates for proportion colonization of AM fungi for each observer. ………………………………………………………………………………. 152

Table 23: Mixed-model ANCOVA of AMF colonization in offspring vs. non-offspring seedlings based on arbuscules only. ………………………………………………….. 154

Table 24: Mixed-model ANCOVA of the survival of seedlings in plots with 4 seedlings only in male and female environments in the field. ………………………………….. 156

Table 25: Mixed-model ANCOVA of the biomass of seedlings in plots with 4 seedlings only in male and female environments in the field. ………………………………….. 157

Table 26: Mixed-model ANCOVA of clipping in male and female environments in the field. ………………………………………………………………………………….. 159

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Table 27: Mixed-model ANCOVA of biomass of non-offspring seedlings only in male and female soil microbial communities in the shadehouse. …………………………... 160

Table 28: Mixed-model ANCOVA of colonization by AM fungi of non-offspring seedlings only in male and female soil microbial communities in the shadehouse. ….. 161

Table 29: Mixed-model ANCOVA of survival of non-offspring seedlings only in male and female environments in the field. ………………………………………………… 162

Table 30: Mixed-model ANCOVA of biomass of non-offspring seedlings only in male and female environments in the field. ………………………………………………… 163

Synthesis: Overview of Research Questions & Summary of Results

Overview of Research Questions

Tropical forests, our most diverse plant communities, have inspired and challenged ecologists (Wilson et al. 2012). In these forests, we search for the mechanisms capable of explaining the coexistence of hundreds or thousands of plant species, all in competition for the same basic resources: light, water, essential nutrients, and a space in the forest (Wright 2002, Leigh et al. 2004). We search for the mechanisms capable of explaining how populations of very rare plant species persist alongside populations of very common plant species, and why a species is common or rare (Chisholm & Muller-

Landau 2011, Comita et al. 2010, Mangan et al. 2010a). We search for the mechanisms capable of explaining the distribution of plants - of answering the simple question of why plants are where we find them (Denslow 1980, Harms et al. 2001, Pearson et al. 2003). In our search, we begin to understand how diversity is maintained in complex natural systems.

It has long been thought that the natural enemies of trees (pathogens, seed predators, and herbivores) are important in determining tropical tree diversity and abundance. Natural enemies reduce plant fitness, impacting the size, structure, and spatial distribution of plant populations (Alexander 2010). Gillett (1962) was the first to hypothesize that when natural enemies are specialists, they help to maintain tree species

diversity by adapting to and predating common species, allowing rare species to escape predation. Janzen (1970) and Connell (1971) expanded on this idea to describe how specialist natural enemies reduce the survival of conspecific seedlings relative to heterospecific seedlings near their host trees (conspecific negative distance-dependent mortality) or when host plant densities are high (conspecific negative density-dependent mortality). By partitioning the regeneration niches of competing plant populations and increasing intraspecific competition relative to interspecific competition, specialist natural enemies can act as a stabilizing mechanism promoting plant species coexistence

(Chesson 2000). Subsequent studies have shown that the Janzen-Connell hypothesis describes patterns of seedling mortality not just in tropical forests, but in a variety of plant communities worldwide, including temperate forests and grasslands (reviewed by

Carson & Schnitzer 2008, Comita et al. 2014). In addition, a recent study found that the strength of negative conspecific density-dependence in tree communities is correlated with the species richness of the community (LaManna et al. 2017).

Soil pathogens can cause negative conspecific distance- and density-dependent seedling mortality through a mechanism called negative plant-soil feedback (Bever et al.

1997). Negative plant-soil feedbacks occur when pathogen communities in the soil change over time in response to nearby plants: specifically, the pathogens that can infect the plants accumulate (Bever 1994, Westover & Bever 2001, Klironomos 2002, Bezemer et al. 2006). Negative plant-soil feedbacks have been documented across a variety of plant communities, including grasslands (Bever 1994, Klironomos 2002, Reynolds et al.

2003), temperate forests (Packer & Clay 2003, McCarthy-Neumann & Ibáñez 2013), and

tropical forests (Mangan et al. 2010a, McCarthy-Neumann & Kobe 2010, Liu et al.

2012). Recent studies indicate that negative plant-soil feedbacks play an important role in determining the diversity and relative abundances of tree species in tropical forests (see

Freckleton & Lewis 2006 for a review; also Mangan et al. 2010a & Bagchi et al. 2014).

The host-specificity of natural enemies is central to the Janzen-Connell hypothesis, but the exact host ranges of many natural enemy species are not known.

Traditionally, natural enemy interactions with tropical trees have been examined at the species level, with the assumption of species-specificity (i.e., each natural enemy species reduces the survival of conspecific seedlings near their host species relative to seedlings of other, non-host species). Within the past decade, experimental studies have provided evidence that natural enemies that specialize more broadly (i.e., on closely related species) (Bagchi et al. 2010, Liu et al. 2012) or more narrowly (i.e., on genotypes within species) (Liu et al. 2015b, Browne & Karubian 2016) can cause Janzen-Connell effects on additional ecological levels. Phylogenetic and intraspecific Janzen-Connell effects suggest that seedling survival is determined not just by species-specific impacts of natural enemies, but by the degree of phylogenetic or genetic relatedness between seedlings and nearby adults. In the words of Janzen (1979), natural enemies do not ‘eat Latin binomials’. Phylogenetic and intraspecific Janzen-Connell effects favor seedlings that are different from established trees and could be important in maintaining phylogenetic diversity within tree communities or genetic diversity within tree species or populations.

Genotype-specific pathogens (i.e., those that vary in their effect on genotypes within

plant species) could be particularly important in maintaining genetic diversity within plant populations.

Association with mutualist soil microbes, such as arbuscular mycorrhizal fungi

(AMF), is an important way that seedlings can offset the negative effects of natural enemies (Liang et al. 2015, Bachelot et al. 2017). Colonization by AMF helps seedlings grow by increasing water and nutrient uptake (Smith & Read 2008) and protects seedlings against natural enemies by activating their host’s defensive pathways (Pozo &

Azcón-Aguilar 2007). Though net-negative impacts of soil microbial communities on seedlings near conspecific adults are more common than net-positive impacts, especially in AMF-associated trees (Mangan et al. 2010, Liu et al. 2012, McCarthy-Neumann &

Ibáñez 2013, Bennett et al. 2017), in at least some tree species, AMF benefits to seedlings are strong enough to balance the negative impact of host-specific pathogens (Liang et al.

2015). In some cases, increases in fitness due to host-specific mycorrhizal fungi outweigh decreases in fitness due to host-specific pathogens and create positive plant-soil feedbacks that promote mono-dominance of plant species (Smith & Reynolds 2012).

Though mycorrhizal fungi are generally thought of as generalists, the host-specificity of mycorrhizal fungi species can vary, as can their impacts on plant fitness (Delavaux et al.

2017). Understanding variation in mycorrhizal fungi association and impact within plant species is important in understanding the role of mycorrhizal fungi in plant regeneration and diversity.

Despite the importance of conspecific negative density- and distance-dependence, it is not known which factors of adult plants or their environments are most important in

contributing to the accumulation of natural enemies near them. Does the adult plant itself attract natural enemies (e.g., with its leaves or root system), or is it the seeds that they produce, or the offspring seedlings that germinate beneath them? Could variation in seed production among adults within species (particularly among males and females of dioecious species) cause the densities of natural enemies near adults to vary, and thus, cause variation in the survival of conspecific seedlings near adults? Variation in the survival of conspecific seedlings near adult plants within species, the factors that could contribute to such variation, and the consequences of such variation for plant diversity are not well understood.

In this dissertation, I aimed to test the following hypotheses:

1) Genotype-specific soil pathogens reduce the performance of offspring seedlings

relative to non-offspring conspecific seedlings in the soil microbial communities

of adult tropical trees.

2) Natural enemies (both pathogens and herbivores) reduce the performance of

offspring seedlings relative to non-offspring conspecific seedlings beneath the

canopies of adult tropical trees in the field.

3) Seed production increases natural enemy densities and reduces conspecific

seedling performance in a) the soil microbial communities of female tropical trees

relative to male trees and b) beneath the canopies of female tropical trees relative

to male trees.

Summary & Synthesis of Results

I found evidence that the soil microbial communities near adults of the tropical tree species Virola surinamensis reduce the growth and colonization by AMF of offspring seedlings relative to non-offspring conspecific seedlings. Seedling growth in these soils was not driven by AMF colonization or soil nutrients, suggesting that highly-specialized soil pathogens are responsible for these reductions. Furthermore, this suggests that the genetic relationship between seedlings and adult conspecifics determines seedling growth and colonization by AMF in conspecific soils: seedlings that were more closely related to the adult performed worse than seedlings in the population that were more distantly related. This pattern of reduced performance of conspecific seedlings in mother soils relative to non-mother conspecific soils was present in most of the maternal seed sources in the study, and was mirrored by reduced performance of offspring seedlings relative to non-offspring seedlings in most of the soil microbial communities in the study. Overall, these patterns reveal the tendency of the soil microbial communities near adult V. surinamensis to favor conspecific seedlings that are more distantly related over conspecific seedlings that are more closely related, suggesting that soil microbial communities help to maintain genetic diversity in the population at local scales.

Plant-soil feedback analysis of seedling growth in a subset of the soil microbial communities in the same experiment revealed a weak, but non-significant, trend towards negative plant soil-feedback in the system. Negative values of plant-soil feedback indicate a tendency of the soil microbial communities in the study to reduce the

performance of the tree’s own offspring relative to non-offspring conspecific seedlings,

(as reflected in the findings described above); however, significantly negative values of plant-soil feedback are necessary to conclude that negative plant-soil feedbacks are occurring within the study population and that soil microbial communities help to prevent the competitive exclusion of genotypes within the study population. Lack of significance of the plant-soil feedback analysis could be the result of small sample size (only a subset of the seedlings, adults, and soils in the larger experiment could be examined in the plant- soil feedback analysis), as the larger experiment indicated a robust trend toward reduced performance of offspring vs. non-offspring seedlings in conspecific soils.

The trend of reduced performance of offspring seedlings relative to non-offspring conspecific seedlings was not present when seedlings were planted beneath the canopies of adult conspecifics in the field: the survival and growth of seedlings of four tropical tree species (including V. surinamensis) were similar between offspring and non-offspring conspecific seedlings near adults of their species. While the shadehouse experiment demonstrated that highly specialized interactions between soil microbes and tropical trees can occur and can impact seedling growth in a way that could maintain genetic diversity, the field experiment revealed that such effects may not be present in all species, or may not be the most important determinants of seedling survival or growth in natural conditions, where seedlings are exposed to all potential natural enemies, variable environments, and other potential stressors (such as drought). Together, my experiments provide evidence that soil microbial communities can be highly specialized and affect conspecific seedling performance in at least some species, but further research is needed

to determine the importance of intraspecific Janzen-Connell effects in determining seedling survival in the field, or the generality of such effects among tropical tree species.

I also tested for variation in conspecific seedling survival, growth, and colonization by AMF in the 1) soil microbial communities of male versus female adults of V. surinamensis in the shadehouse and 2) environments near male versus female adults of V. surinamensis in the field. In the shadehouse, seedlings were exposed only to the soil microbes near males and females, with other environmental variables controlled. In the field, seedlings were exposed to all potential natural enemies and variable environmental conditions. I did not find evidence of variation in conspecific seedling survival or growth near males versus females in the field, or in seedling growth or colonization by AMF in male versus female soil microbial communities in the shadehouse. This suggests that male and female soil microbial communities and environments do not differ in this species in a way that is meaningful for determining the performance of conspecific seedlings, and that seed production does not cause large variation in interactions between natural enemies and seedlings. In addition, the growth and colonization by AMF of seedlings grown in the soil microbial communities of females that varied in seed production also did not differ. This suggests that, if seed production does increase natural enemy densities, that such changes do not meaningfully alter the interactions of conspecific soil microbial communities with seedlings. Overall, male and female conspecific sites were similar in their suitability for conspecific seedlings, as were female soils that varied in seed production, indicating that seed production is not an important factor determining seedling survival near conspecific adults of this species.

Intraspecific variation in patterns of seedling survival near conspecific adults is a potentially important, but underexplored topic in plant ecology. Little is known about the factors that contribute to the accumulation of natural enemies near adult plants, or the factors that determine the extent to which natural enemies reduce the survival of conspecific seedlings near these adults. My experiments addressed one potential factor that could be important in determining the accumulation of natural enemies near adult plants and resulting patterns of conspecific seedling survival, seed production, and found that seed production did not cause variation in patterns of conspecific seedling survival.

This suggests that other factors, such as tree size, neighborhood, conspecific seedling neighbors, or abiotic variables, are more important in determining natural enemy accumulation and patterns of conspecific seedling survival. In addition, my experiments suggest that soil microbial communities can sometimes specialize within plant species and influence the performance of conspecific seedlings near adults in a way that is determined by the degree of genetic relatedness between the seedlings and the adults.

This indicates that genotype-specific soil pathogens could be an important factor structuring, and potentially maintaining, genetic diversity within tropical tree species and populations. Going forth, I suggest future studies examine: 1) variation in pathogen resistance genes within tropical tree species, and whether genetic relatedness determines seedling recruitment near conspecific adults; 2) the level of host-specificity of natural enemy species, especially genotype-specificity within tropical tree species and populations, and the genetic basis of this specificity; and 3) variation in natural enemy composition and abundance near adults within species. By looking beyond species to

examine intraspecific variation in interactions between plants and natural enemies, we can begin to understand the genetic basis of these interactions and how these interactions shape genetic and species diversity and abundance in plant communities.

Chapter 1: Soil Microbes reduce Growth of Offspring vs. Non-offspring

Conspecific Seedlings: Experimental Evidence of a Janzen-Connell

Effect within a Tropical Tree Population

Abstract

The Janzen-Connell hypothesis describes how soil microbes that differentially affect plant species, such as pathogens and mycorrhizal fungi, can promote species diversity in plant communities by accumulating near their host plants and limiting the recruitment of conspecific seedlings. In agricultural and model plant systems, it is well- known that microbes can also specialize within plant species (i.e. on genotypes), but the impact of such highly specialized microbes on diversity within wild plant species has rarely been examined. Here we show that soil microbial communities from beneath parent trees have stronger negative effects on a tree’s own offspring than on other seedlings in the population. This indicates that soil microbes can specialize within plant species in natural systems and may play a role in driving ecological and evolutionary dynamics in wild plant populations and communities. In a shadehouse experiment in

Panama, we grew seedlings of the tropical tree, Virola surinamensis (Myristicaceae), in the soil of either their own parent tree or a different parent in the population, and found that seedlings grown in the soil from beneath their own parent had lower biomass than

those grown in the soil of a different parent. Colonization by arbuscular mycorrhizal

(AM) fungi was also lower in the seedlings grown in their own parent’s soil than in the seedlings grown in the soil of a different parent; however, there was no relationship between colonization by AM fungi and biomass in the seedlings, suggesting that AM fungi are not driving the reductions in biomass in offspring relative to other seedlings.

Our results suggest that highly specialized soil pathogens reduce the growth of seedlings that are closely related to nearby conspecifics, which could promote genetic diversity within plant populations via an extension of the Janzen-Connell hypothesis. Future studies examining the specificity of soil microbes at the genotype level within wild plant populations would help confirm this hypothesis and expand our understanding of how specialist microbes maintain diversity at multiple levels in wild plant communities.

Introduction

Microbes that impact the fitness of wild plants (e.g., pathogens and mutualists) play an essential role in driving patterns of diversity and abundance in plant populations and communities worldwide (Gilbert 2002, Alexander 2010). The Janzen-Connell hypothesis (Janzen 1970, Connell 1971) posits that specialized pathogens promote plant species diversity by limiting the regeneration of their host plants when and where they are common, allowing rare species to persist (Adler & Muller-Landau 2005). Specifically, species-specific pathogens accumulate in the environment near their host plants

(distance-dependence) or when host plant densities are high (density-dependence) and suppress the survival or growth of additional conspecifics, allowing the regeneration of

non-host or rare plant species at a site. The demographic patterns predicted by Janzen

(1970) and Connell (1971) have been documented in numerous plant communities worldwide (e.g., tropical and temperate forests, grasslands) (reviewed by Comita et al.

2014), with accumulating evidence suggesting that these patterns are often caused by host-specific pathogens in the soil (Augspurger & Kelly 1984, Packer & Clay 2003, Bell et al. 2006, Bagchi et al. 2010, Mangan et al. 2010a, McCarthy-Neumann & Kobe 2010,

Liu et al. 2012, Liu et al. 2015a, Liang et al. 2016).

The Janzen-Connell hypothesis focuses on the role of species-specific pathogens in maintaining plant species diversity, but pathogens could also help maintain genetic diversity within their host plant populations if their impacts vary within the plant population (i.e., among host genotypes). Agricultural and model plant studies show that rapid, local adaptation of pathogens to plant resistance (Mursinoff & Tack 2017) can result in highly specialized pathogen races that differentially impact genotypes within a plant species (reviewed by Laine et al. 2011 and Whitham et al. 2012), but genotype- specific pathogens have seldom been studied in the context of the Janzen-Connell hypothesis (but see Augspurger & Kelly 1984, Reinhart & Clay 2009, Liu et al. 2015b,

Akaji et al. 2016). Genotype-specific pathogens could promote genetic diversity in their host populations by producing Janzen-Connell effects within the population (see Figure 1 for a conceptual diagram). By accumulating in the soil near plants with genotypes they can infect and limiting the recruitment of the plant’s offspring (who share the susceptible genotype) relative to other, less-susceptible seedlings in the population, genotype-specific pathogens could contribute to an intraspecific extension of the plant-soil feedback

framework (Bever et al. 1997). Negative plant-soil feedbacks define the criteria necessary for soil pathogens to promote stable coexistence of competing plant species in a community, and are the mechanism by which soil pathogens produce Janzen-Connell demographic patterns. Similarly, negative plant-soil feedbacks caused by genotype- specific pathogens could lead to Janzen-Connell-like promotion of genetic diversity within plant populations and promote the coexistence of competing genotypes within plant populations and/or species.

Figure 1: Conceptual diagram of how genotype-specific soil pathogens could promote genetic diversity in plant populations.

Despite the potential for genotype-specific pathogens to produce Janzen-Connell effects that promote genetic diversity in plant populations and/or species, this hypothesis has rarely been tested in wild plants. A recent study provided the first evidence of intraspecific Janzen-Connell effects by showing that the soil microbial communities from

beneath parent plants of two subtropical tree species promote genetic diversity within their species by producing stronger negative effects on seedlings from their same population relative to seedlings from genetically distant populations (Liu et al. 2015b).

Variation in pathogen impacts can be even greater within plant populations than among plant populations (reviewed by Tack et al. 2012), but it is unknown whether pathogens can help promote the recruitment of genetically diverse seedlings within a single wild plant population. Only a handful of ecological studies have tested whether seedling survival within a plant population is determined by the genetic relatedness of seedlings to nearby conspecific adults, with mixed results. A field experiment with a population of a tropical palm in Ecuador showed that seedlings with rarer genotypes were more likely to survive near conspecific adults than seedlings with more common genotypes (Browne &

Karubian 2016), but could not attribute this pattern to pathogens specifically. On the other hand, an observational study with a temperate tree population in Japan did not find a relationship between genetic relatedness to nearby adults and seedling survival in the field (Akaji et al. 2016). A field experiment with seedlings of three parent trees in a tropical tree population in Panama showed some evidence that genetic relatedness leads to higher pathogen impacts by showing that disease levels higher for seedlings of one of the three trees near their own parent tree than near the other two conspecific adults

(Augspurger & Kelly 1984). The hypothesis that genetic relatedness between conspecific seedlings and adults determines seedling survival remains to be tested in a great many plant species and populations. Intraspecific variation in seedling recruitment due to

highly specialized pathogens is a largely underexamined topic that could help us to understand how genetic diversity is maintained in wild plant populations and species.

Mutualists, such as mycorrhizal fungi, also play an important role in the processes maintaining plant species diversity (Bever et al. 2002, Hood et al. 2004, Mangan et al.

2010b, Smith & Reynolds 2012, Bachelot et al. 2015, Liang et al. 2015, Bachelot et al.

2017). Mycorrhizal fungi provide plants with a growth advantage (Smith & Read 2008) and can protect plants against pathogens by inducing plant defenses (Pozo & Azcón-

Aguilar 2007) or by providing physical barriers against pathogen colonization (e.g., the sheath of ectomycorrizhal fungi) (Newsham et al. 1995), among other benefits (Delavaux et al. 2017). Though mycorrhizal fungi are generally thought of as generalists, like pathogens, mycorrhizal fungi can structure plant species diversity by differentially benefitting their host plants (Bever 2002, Mangan et al. 2010b, Smith & Reynolds 2012,

Liang et al. 2015, Bachelot et al. 2017). Mycorrhizal fungi can reduce plant diversity by facilitating the recruitment of conspecific plants near their hosts (Hood et al. 2004, Liang et al. 2015) or promote plant diversity by providing greater benefits to their hosts where they are rare (Bever 2002, Bachelot et al. 2017). Mycorrhizal fungi can also vary in the benefits they provide to genotypes within a host plant species (Ronsheim & Anderson

2001), but the patterns of genetic and/or species diversity produced by highly specialized mycorrhizal fungi are only beginning to be explored.

We conducted a shadehouse experiment with the tropical tree Virola surinamensis in Panama to determine whether soil microbial communities (pathogens and/or mutualists) vary in their impact on seedlings from the same population in a way that

could promote genetic diversity within the population. We hypothesized that soil microbial communities near parent trees in the population specialize over time on the genotype of the tree and therefore limit the recruitment of the tree’s own offspring relative to non-offspring seedlings in the population. This would favor the recruitment of seedlings that are genetically distant from the trees they are near (i.e., seedlings with locally rare genotypes) and could help to maintain genetic diversity within the tree population. To test this hypothesis, we compared the seedling biomass and association with arbuscular mycorrhizal fungi of 139 seedlings from 11 parent trees in the same population when grown for eight months in either the soil microbial community of their own parent tree (i.e., offspring seedlings) or the soil microbial community of a tree in the population that is not their parent (i.e., non-offspring seedlings). We also calculated plant-soil feedbacks based on seedling growth for the subset of five seedling cohorts and soil microbial communities in the experiment for which fully-reciprocal data were available (i.e., for which seedlings from each of the five parents were planted in soil from each of the five parents). Negative values of plant-soil feedback are the criteria necessary to prevent competitive exclusion and to maintain stable coexistence of competing genotypes within the plant population. In addition, we tested whether colonization by AM fungi and/or soil nutrients explains seedling biomass.

Methods

Study site and species

We conducted our study on Virola surinamensis (Rol. ex Rottb.) Warb.

(Myristicaceae), a tropical canopy tree species, on Barro Colorado Island (BCI), Republic of Panama (9°09’ N, 79°51’ W). BCI is a 15.6 km2 lowland, tropical moist forest (Croat

1978). BCI is seasonally dry, receiving an average of 2623 mm of rain per year, punctuated by a distinct dry season (Windsor 1990). Virola surinamensis is a dioecious species native to tropical and subtropical wet lowland forests across Central America and

Amazonia, and is relatively common on BCI (Croat 1978). On BCI, adults are most commonly found on slopes and near streams (Harms et al. 2001). Virola surinamensis adults are spatially aggregated (Condit et al. 2000, Riba-Hernández et al. 2014), and were found to have random sex distributions and a uniform sex ratio in Costa Rica (Riba-

Hernández et al. 2014). On BCI, Flowering of V. surinamensis peaks in the dry season (~

January) and seed production peaks in the following wet season (~ July) (Zimmerman et al. 2007). In Brazil, flowers were found to be pollinated by two species of flies in the

Syrphidae family (Copestylum sp. and Erystalys sp.; Jardim & Mota 2007). Seeds are large (~2 cm), borne singly inside a woody capsule, and are surrounded by a nutritious, red aril that attracts several species of large birds, such as chestnut-mandibled and keel- billed toucans (Ramphastos swainsonii and Ramphastos sulfuratus, respectively), crested guans (Penelope purpurascens), slaty-tailed trogons (Trogon massena), rufous motmots

(Baryphthengus martii), and collared aracaris (Pteroglossus torquatus), as well as spider monkeys (Ateles geoffroyi), as primary seed dispersers (Howe & Vande Kerckhove

1981). On average, 62 + 5% of the seeds produced by a maternal tree are dispersed away by animals, while the remaining 37% are dropped or regurgitated under the tree (Howe &

Vande Kerckhove 1981). Fruit crop size varies by tree, but hundreds to thousands of seeds can be dropped below maternal crowns (Howe et al. 1985; Eck, unpublished data).

Insects and mammals kill 99.2% of seeds and/or seedlings near maternal trees within the first 12 weeks after germination (Howe et al. 1985). Virola surinamensis seedlings are shade-tolerant (Howe 1990), drought-sensitive (Fisher et al. 1991), and are more likely to survive if dispersed >20 m away from the maternal tree (Howe et al. 1985). Obligate outcrossing, animal seed dispersal, and high mortality of offspring seeds and seedlings near mother trees all likely contribute to the weak, but significant, spatial genetic structure observed in a population of V. surinamensis in Costa Rica: relatedness decreases as distance between adult trees increases (Riba-Hernández et al. 2014).

Shadehouse experiment: mother vs. other conspecific soil microbial communities

We collected 2,854 seeds from the ground beneath the canopy of 28 fruiting V. surinamensis during the peak of the species’ fruiting event (in June and July) on BCI in

After collection, seeds were surface sterilized (10% bleach for 2 min., rinse, 70% ethanol

for 2 min., rinse), air dried, planted in germination trays containing autoclaved BCI soil in the shadehouse, and kept well-watered and shaded under two layers of 80% shadecloth. Seed germination rates were low (~11%). The parent trees yielding > 6 seedlings healthy in appearance one month after germination were included in the experiment, for a total of 11 parent trees and 144 experimental seedlings. A spatial map of the location of the 11 trees on BCI is provided in Figure 2. Spatial distances between pairs of parent trees ranges from ~30 m to ~ 2 km. Based on research at another site, pairs at the lower end of this range are more likely to be relatives (Riba-Hernández et al. 2014), but the level of relatedness of the parent trees and seedlings in this study is not known.

The 11 parent trees were large, ranging from 50 - 100 cm DBH (diameter at breast height); the minimum reproductive size for V. surinamensis is ~30 cm DBH (Riba-

Hernández et al. 2014).

Figure 2: A spatial map of the location of the 11 maternal trees in the ‘mother vs. other’ shadehouse experiment on BCI. The location of each maternal Virola surinamensis seed source and soil microbial inoculum source in the experiment (labelled V1 through V11). Satellite image obtained from Google Maps ® 2017.

We collected a sample of the soil microbial community beneath the canopy of each of the parent trees in the experiment to use as inocula, for a total of 11 inocula (one per parent tree). To create inoculum for each parent tree, we collected soil at a depth of

10 cm from three randomly-selected points within 3 m of the trunk, then coarsely sieved, combined, and homogenized the soil from the three points. All soil microbial community samples were collected in September 2014 and all inocula were created and used in the experiment within two days after field collection.

Within each of the 11 maternal cohorts, seedlings were assigned at random to one of two experimental groups: 'offspring’ or ‘non-offspring’, such that half of each cohort was assigned to each group. Each seedling in the offspring group (n = 71 seedlings) was planted in the soil microbial inoculum of its own parent tree. Each seedling in the non- offspring group (n = 68 seedlings) was assigned to one of the experimental trees that was not its parent, and planted in the soil microbial inoculum of that tree. Thus, each seedling was planted in the soil microbial community of only one adult in the experiment (either its own parent, or an adult that was not its parent). The experimental design also featured a nested 6 x 6 plant-soil feedback sub-experiment with the six parents that provided sufficient numbers of seedlings. In the plant-soil feedback sub-experiment, seedlings from each of the six mothers were assigned to soil microbial inocula from each of the six mothers in a reciprocal design. Which seedling in the cohort was assigned to which adult was determined randomly. Sample sizes for each combination of maternal seed source and soil source in the experiment are listed in Table 1.

Table 1: Sample sizes for each combination of maternal seed source and soil microbial source in the ‘mother vs. other’ shadehouse experiment. The number of seedlings planted in each combination of seed source and soil source at the beginning of the experiment. Seedlings in the offspring group are in bold (n = 71 seedlings), the remaining seedlings are in the non-offspring group (n = 68 seedlings). Seed and soil sources F1 through F6 comprise the six seed and soil sources in the plant-soil feedback sub-experiment. Dashes represent lack of replicates.

Each seedling was transplanted at ~1 month of age in to its own 2 L pot containing 20% by volume of soil microbial inoculum from its assigned tree and 80% by volume of a common planting medium (a steam-sterilized 1:1 mixture of BCI soil and sand). Relatively small volumes of soil microbial inoculum were used to minimize the impact of any potential variation in soil nutrients among inocula. Each pot was randomly assigned to one of four benches in a shadehouse on BCI. Each bench was covered with two layers of 80% shade cloth (to mimic a shady understory) and was shielded from rainfall with a roof of clear plastic lining. Initial stem height, leaf number, and leaf area

(length and width) of each seedling was measured immediately after transplant. Seedlings

remained in their experimental treatments for eight months, during which time seedling survival and growth (stem height, leaf number, and leaf length and width) were measured periodically (every 2-4 weeks), and each seedling was watered three times per week (see

Figure 3 for a photograph of the set-up in the shadehouse). After eight months, the surviving seedlings were harvested (i.e., gently dug from their pots and their roots washed to remove soil) to obtain measurements of final oven-dried biomass, stem height, and leaf area, and to collect fine root samples for the AM fungi analysis. Fine root samples were stored in 70% ethanol and refrigerated until being shipped to Yale

University (USA) for AM fungi quantification.

Figure 3: A photograph of the ‘mother vs. other’ shadehouse experiment. The photograph shows V. surinamensis seedlings growing individually in pots containing the soil microbial community of either their own parent tree or a different parent in the population.

Quantification of colonization by AM fungi

To quantify colonization by AM fungi in the seedlings, we used a magnified root intersect method (McGonigle et al. 1990). To prepare roots for quantification, we used a modified root clearing and staining protocol (Giovannetti and Mosse 1980; McGonigle et al. 1990; Vierheilig et al. 2005; INVAM 2015). For each seedling, we cleared a random

0.2 mg subsample of the seedling’s fine root mass in 10% bleach, 10% KOH, and 1%

HCL. We then stained the subsamples using direct blue stain and mounted them on glass slides using lactic acid. We quantified the colonization of AM fungi in the subsamples at

200x magnification under a compound light microscope by recording the presence or absence of visible AM fungi-distinctive structures (hyphae, vesicles, arbuscules, or arbuscular coils) for a minimum of 35 root intersects per seedling (see Figure 4 for a micrograph of the structures quantified). We obtained high-quality AM fungi colonization data for 112 of the 139 surviving experimental seedlings. We calculated AM fungi colonization for each of these seedings as the proportion of root intersects quantified in the subsample that contained any visible AM fungi structure. We also calculated the proportion of root intersects quantified that contained visible arbuscules only, as a more conservative estimate of colonization by AM fungi.

Figure 4: A micrograph of AM fungi structures in the root of an experimental seedling. A photograph taken at 200x magnification shows examples of the four AM fungi structures quantified in the AM fungi study (hyphae, arbuscules, vesicles, and coils) stained in blue inside the roots of an experimental seedling.

Quantification of nutrients in the soil microbial inocula

At the time of harvest, a sample of the soil surrounding the roots of each surviving seedling was collected and refrigerated for one year. Then, for each of the 11 soil sources in the experiment, a small amount of soil from each of the samples in the soil source was combined and homogenized to create one 50 g soil sample for each of the 11 soil sources.

These 11 soil samples were taken to the Soils Lab at Smithsonian Tropical Research

Institute (Panama) for nutrient quantification. Soil pH (in water and CaCl2), soil phosphorous (total P mg/soil kg and Bray 1-P mg/soil kg), soil organic matter (loss on

ignition), soil carbon (% total C), soil nitrogen (% total N), and soil C:N ratio were quantified for each of the 11 soil sources in the experiment.

Statistical analysis

We analyzed the effect of the experimental treatment group (i.e., offspring vs. non-offspring seedlings) on seedling total dry biomass at harvest using a linear mixed- effects model. We focused on growth because survival was high in the experiment

(96.5%, 139 out of 144 seedlings). Dead seedlings were not included in the growth analysis. Maternal seed source, soil microbial inoculum source, and shadehouse bench were included as random effects in the model. To account for the potential impact of variation in the size of the seedlings at the beginning of the experiment, we also included an estimate of initial seedling dry biomass as a covariate in the model. Initial dry biomass was estimated for each experimental seedling based on height at the time of transplant using an allometric linear regression model based on measurements of height and biomass of a randomly harvested sample of the potential experimental seedlings (F1,42 =

338.1, p < 2.2e-16, R2 = 0.887).

We also analyzed the effect of AM fungi colonization on seedling total dry biomass at harvest using a separate linear mixed-effects model. As above, estimated initial seedling dry biomass was included as a covariate, and maternal seed source, soil microbial inoculum source, and shadehouse bench were included as random effects. We standardized the variable of proportion colonization by AM fungi used in this model to

correct for differences in AM fungi quantification between the two observers in the experiment (standardization of this variable is described in Appendix A).

We also analyzed the effect of soil nutrients on seedling total dry biomass at harvest. First, we conducted a principal components analysis (PCA) including all soil nutrient variables for which data was available. PCA revealed that total phosphorous is the best indicator of overall soil nutrient status (Appendix B, Figure 38). We then analyzed the effect of soil nutrients (total phosphorous) on seedling dry biomass at harvest using a linear mixed-effects model. In this model, we included initial seedling biomass as a covariate and maternal seed source, soil microbial inoculum source, and shadehouse bench as random effects.

We also analyzed plant-soil feedback based on seedling biomass among five of the six maternal seed sources and soil microbial communities in the plant-soil feedback sub-experiment (one seed/soil source was dropped from analysis of the sub-experiment due to missing biomass values from dead seedlings). To do this, we first constructed a linear mixed effects model based on the growth (log-transformed biomass) of the seedlings that survived the experiment. In this model, maternal seed source, soil microbial inoculum source, and the interaction between the two were included as fixed effects, along with a log-transformed estimate of initial seedling biomass as a covariate.

Shadehouse bench was also included as a random effect. Within the ‘maternal seed source x soil microbial inoculum source’ interaction, we used the multcomp package

(Hothorn et al. 2008) to conduct a priori contrasts that isolated the strength and direction of the interaction between maternal seed source and soil microbial inoculum source for

each possible pair of parent trees (i.e., pairwise feedbacks, calculated with Equation 1, as described in Bever et al. 1997). Pairwise contrasts compared the relative growth response of seedlings when associated with soil microbial inoculum from their own parent tree

(αA) versus from a non-parent tree (αB), relative to how seedlings of B responded across soil microbial inocula from A (βA) and from B (βB). Then, average feedback values for each source were obtained by calculating the average pairwise feedback value for all other seed sources in the soil source ((β,C,D,E)A), the average pairwise feedback value for the seed source in all other soil sources in the subset (α(B,C,D,E)), and the average pairwise feedback value for all other seed sources ((β,C,D,E)(β,C,D,E)) in all other soil sources, and using these values in place of the latter three terms of Equation 1.

We also analyzed the effect of the experimental treatment group (i.e., offspring vs. non-offspring seedlings) on the proportion of AM fungi colonization in the surviving seedlings at the end of the experiment using a generalized linear mixed-effects model with binomial errors. As above, estimated initial seedling dry biomass was included as a covariate, and maternal seed source, soil microbial inoculum source, and shadehouse bench were included as random effects in the model. To account for potential variation between the two researchers that quantified seedling AM fungi colonization, we included observer as a fixed effect in the model. We also tried a version of this model with identical terms but using colonization proportion of arbuscules only as the response 29

variable, and compared the results of this more conservative model to the model of colonization including all structures.

We also analyzed the effect of soil nutrients (total P) on seedling AMF colonization (proportion of fine roots colonized) at harvest using a generalized linear mixed-effects model. In this model, we included log-transformed initial seedling biomass as a covariate, observer as a fixed effect, and maternal seed source, soil microbial inoculum source, and shadehouse bench as random effects.

All mixed-effects analyses in our study were performed using the lme4 package

(Bates et al. 2015) in the R environment (R Core Team 2017). R2 values for all mixed- effects analyses were obtained with the MuMIn package (Barton 2016). P-values for mixed-model predictors were obtained with the lmerTest package (Kuznetsova et al.

2016). ANOVA tables for the mixed-effects models were obtained with the afex package

(Singmann et al. 2017).

Results

Seedling biomass

Most seedlings grew substantially during the experiment: total dry biomass at the end of the experiment was 2.56 g on average, while average total dry biomass at the beginning of the experiment was estimated at 0.38 g. Final biomass was significantly lower for seedlings growing in their own parent’s soil microbial community (i.e., offspring seedlings) relative to seedlings growing in the soil microbial community of a different parent in the population (i.e., non-offspring seedlings) (Table 2 & Figure 5).

This pattern of growth was observed for the majority of maternal seed sources and soil microbial communities: eight out of the 11 seed sources in the experiment experienced lower growth in their parent’s soil microbial community relative to the soil microbial community of a non-parent conspecific, and growth was lower in offspring relative to non-offspring seedlings in four out of the six soil microbial communities tested (Figure

6). Seedling biomass was not affected by levels of AM fungi colonization in roots (Table

3 & Figure 7) or by soil nutrients (Table 4).

Table 2: Mixed-model ANCOVA of biomass in offspring vs. non-offspring seedlings in the ‘mother vs. other’ shadehouse experiment. ANCOVA table of the predictors of final dry biomass of offspring vs. non-offspring seedlings in conspecific soil microbial communities at the end of the 8-month soil microbial inoculation shadehouse experiment (n = 139 seedlings). Experimental treatment = whether a seedling was the offspring or a non-offspring of the adult tree whose soil microbial community it was grown in. Initial biomass = a seedling’s initial dry biomass (estimated). Fixed effects summary includes coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), t-score (t = β/SE(β)), F-value (F), and significance level (p). The intercept represents seedlings in the offspring treatment. Random effects summary includes variance and standard deviation of all random effects (i.e., 11 maternal seed sources, 11 soil microbial inoculum sources, and 4 shadehouse benches).

Fixed Effects β SE(β) df t F p

Intercept 0.82 0.38 5.89 2.13 NA 0.08

Non-offspring 0.29 0.12 124.32 2.35 5.53 0.02 seedlings

Initial Biomass 3.96 0.45 129.38 8.85 78.33 5.77e-15

Random Effects Var. SD Maternal Seed Source 0.090 0.300 Soil Microbial Inoculum Source 0.027 0.166 Bench 0.401 0.633 Residual 0.502 0.708

Figure 5: Biomass of offspring vs. non-offspring seedlings in conspecific soil microbial communities in the ‘mother vs. other’ shadehouse experiment. The biomass of offspring seedlings (in their own parent’s soil microbial community) vs. non-offspring seedlings (in a non-parent conspecific soil microbial community) at the end of the 8-month shadehouse experiment (n = 139 seedlings). A linear mixed-effects model revealed that seedlings in the offspring group had significantly reduced biomass relative to seedlings in the non-offspring group (differing letters indicate p < 0.05). Whisker lengths are + 1.5 * the interquartile range (IQR).

Figure 6: Biomass of seedlings across 11 maternal seed sources and 11 soil microbial communities in the ‘mother vs. other’ shadehouse experiment. Panel A: The final biomass of seedlings from 11 maternal seed sources in parent and non-parent soil microbial communities (n = 139 seedlings). Mean seedling biomass was lower in the parent’s soil microbial community relative to non-parent conspecific soil microbial communities in eight out of the 11 maternal seed sources. Panel B: The final biomass of offspring vs. non-offspring conspecific seedlings in 11 soil microbial communities (n = 139 seedlings). Mean seedling biomass was lower in offspring seedlings relative to non- offspring conspecific seedlings in four out of the six soil microbial communities for which data was available (NAs represent missing data). Whisker lengths are + 1.5*IQR.

Table 3: Mixed-model ANCOVA of the impact of colonization by AM fungi on seedling biomass in the ‘mother vs. other’ shadehouse experiment. ANCOVA table of colonization by AM fungi and initial biomass as predictors of final biomass of experimental seedlings in conspecific soil microbial communities at the end of the 8- month soil microbial inoculation shadehouse experiment (n = 112 seedlings). AMF colonization = the proportion of a seedling’s fine roots colonized by AM fungi (standardized to account for differences between observers). Initial biomass = a seedling’s initial dry biomass (estimated).

Fixed Effects β SE(β) df t F p

Intercept 1.32 0.43 5.32 3.04 NA 0.03

AMF Colonization -0.11 0.34 103.35 -0.33 0.11 0.74

Initial Biomass 3.22 0.46 99.81 6.96 48.37 3.73e-10

Random Effects Var. SD Maternal Seed Source 0.107 0.328 Soil Microbial Inoculum Source 0.031 0.176 Bench 0.545 0.738 Residual 0.370 0.609

Figure 7: Seedling growth as a function of AMF colonization in conspecific soil microbial communities in the ‘mother vs. other’ shadehouse experiment. Total seedling biomass as a function of proportion AMF colonization for seedlings grown in conspecific soil microbial communities at the end of the 8-month shadehouse experiment (n = 112 seedlings). Seedlings in the ‘offspring’ treatment are shown as blue dots; seedlings in the ‘non-offspring’ treatment are shown as pink dots. A linear mixed-effects model revealed that AMF colonization did not have a significant effect on seedling biomass (p = 0.76).

Table 4: Seedling biomass as a function of soil nutrients in the ‘mother vs. other’ shadehouse experiment. ANCOVA table of soil nutrients as a predictor of the biomass of experimental seedlings in conspecific soil microbial communities at the end of the 8- month soil microbial inoculation shadehouse experiment (n = 139 seedlings). Soil nutrients = the total phosphorous (mg P/kg soil) of the soil microbial inoculum a seedling was grown in. Initial biomass = a seedling’s estimated initial dry biomass.

ANCOVA df F p

Soil nutrients 1, 6.31 0.12 0.74

Initial Biomass 1, 129.51 78.75 < 0.001 ***

Plant-soil feedback based on seedling biomass

A plant-soil feedback analysis of the growth of the 83 V. surinamensis seedlings in the plant-soil feedback subset showed that average plant-soil feedback values were negative for all five seed/soil sources tested, but none of the values were negative enough to vary statistically from zero (Table 5, Figure 8, & Figure 9). This resulted in a negative but statistically neutral system-wide plant-soil feedback value for the population (Table 6).

Table 5: Average plant-soil feedback among mother trees in the ‘mother vs. other’ shadehouse experiment. A priori contrasts of the average plant-soil feedback strength and direction based of the five parent trees in the plant-soil feedback subset based on seedling biomass at the end of the 8-month shadehouse experiment (n = 83 seedlings). Summary includes plant-soil feedback estimates (β), standard errors (SE), Wald’s z-score (z = β/SE(β)) and significance level (p) for all linear hypotheses.

Plant-soil feedback Source SE(β) z p (β)

F1 -0.14 0.18 -0.81 0.92

F2 -0.26 0.19 -1.37 0.56

F4 -0.16 0.17 -0.90 0.87

F5 -0.11 0.16 -0.69 0.95

F6 -0.18 0.18 -0.99 0.83

Table 6: Mixed-model ANCOVA of system-wide plant-soil feedback in the ‘mother vs. other’ shadehouse experiment. ANCOVA table of the predictors of the biomass of seedlings in the plant-soil feedback subset at the end of the 8-month soil microbial inoculation shadehouse experiment (n = 83 seedlings). Seed source x soil source = the interaction between maternal seed source (parent tree) and soil microbial inoculum source. Initial biomass = a seedling’s estimated initial dry biomass. The p-value for the seed source x soil source interaction gives the significance of the plant-soil feedback value for the system. The p-value of 0.20 indicates that the system did not experience statistically significant levels of plant-soil feedback.

df F p

Seed Source x Soil Source 24, 54.38 1.32 0.20

Initial Biomass 1, 56.94 18.43 < 0.001

Figure 8: Plant-soil feedback among mother trees in the ‘mother vs. other’ shadehouse experiment. Average plant-soil feedback (values of Is from Equation 1) of seedling biomass mediated by soil microbial communities among the five parent trees in the plant-soil feedback subset (n = 83 seedlings). Error bars are 95% family-wise confidence intervals.

Figure 9: Plant-soil feedback of conspecific seedlings.

Biomass of seedlings of both the target mother and other conspecifics when grown in soil microbial inoculum collected from the target mother versus other conspecifics. Biomass values for other conspecific soils are averaged across seedlings from each group in the non- target conspecific soils. Each graph targets one of the five conspecific mothers (M1, M2, M4, M5, or M6) in the plant-soil feedback sub-experiment. Error bars are + one standard error of the mean. 40

Seedling colonization by AM fungi

We found that colonization by AM fungi was lower in seedlings growing in their own parent’s soil microbial community (i.e., offspring seedlings) relative to seedlings growing in the soil microbial community of a non-parent tree in the population (i.e., non- offspring seedlings) (Table 7 and Figure 10). Seven of the 11 seedling cohorts in the experiment had lower AMF colonization in their own parent’s soil microbial community relative to the soil microbial community of a non-parent conspecific, and AMF colonization was lower in offspring relative to non-offspring seedlings in three out of the six soil microbial communities tested (Figure 11). These results were similar between the model of colonization proportion based on all AM fungal structures and model of colonization proportion based on arbuscules only (Appendix A: Table A3). In addition, seedling proportion colonization by AM fungi was not affected by soil nutrients (Table

8).

Table 7: Mixed-model ANCOVA of colonization by AM fungi in offspring vs. non- offspring seedlings in the ‘mother vs. other’ shadehouse experiment. ANCOVA table of the predictors of colonization by AM fungi of offspring vs. non-offspring experimental seedlings in conspecific soil microbial communities at the end of the 8-month soil microbial inoculation shadehouse experiment (n = 112 seedlings). Experimental treatment = whether a seedling was the offspring or a non-offspring of the adult tree whose soil microbial community it was grown in. Initial biomass = a seedling’s initial dry biomass (estimated). Observer = which of two researchers quantified colonization by AM fungi in the seedling. Fixed effects summary includes coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), z-score (z = β/SE(β)), F-value (F), and significance level (p). The intercept represents seedlings in the offspring treatment. Random effects summary includes variance and standard deviation of all random effects (i.e., 11 maternal seed sources, 11 soil microbial inoculum sources, and 4 shadehouse benches).

Fixed Effects β SE(β) z df F p Intercept -2.52 0.42 -5.95 NA NA 2.76e-09

Non-offspring Seedlings 0.43 0.06 6.96 101.50 47.47 3.41e-12

Initial Biomass 1.54 0.22 7.11 96.57 92.33 1.16e-12

Observer 0.81 0.06 13.15 105.34 173.37 < 2e-16

Random Effects Var. SD Maternal Seed Source 0.216 0.464 Soil Microbial Inoculum Source 1.104 1.051 Bench 0.197 0.444

Figure 10: Colonization by AM fungi in offspring vs. non-offspring seedlings in conspecific soil microbial communities in the ‘mother vs. other’ shadehouse experiment. The proportion of colonization by AM fungi in the roots of offspring seedlings (in their own parent’s soil microbial community) vs. non-offspring seedlings (in the soil microbial community of a non-parent conspecific) at the end of the 8-month shadehouse experiment (n = 112 seedlings). A generalized-linear mixed effects model revealed that seedlings in the offspring group had significantly reduced colonization by AM fungi relative to seedlings in the non-offspring group (differing letters indicate p < 0.05). Whisker lengths are + 1.5*IQR.

Figure 11: Colonization by AM fungi of seedlings across 11 maternal seed sources and 11 soil microbial communities in the ‘mother vs. other’ shadehouse experiment. Proportion colonization by AM fungi of seedlings from 11 maternal seed sources in 11 conspecific soil microbial communities (n = 112 seedlings). AMF colonization was lower in the parent’s soil microbial community relative to non-parent conspecific soil microbial communities in seven out of the 11 seed sources (Panel A). Seedling colonization by AM fungi was lower in offspring seedlings relative to non-offspring conspecific seedlings in three out of the six soil microbial communities for which data was available (Panel B; NAs represent missing data). Whisker lengths are + 1.5*IQR.

Table 8: Colonization by AM fungi as a function of soil nutrients in the ‘mother vs. other’ shadehouse experiment. ANCOVA table of soil nutrients as a predictor of the AMF colonization of experimental seedlings in conspecific soil microbial communities at the end of the 8-month soil microbial inoculation shadehouse experiment (n = 112 seedlings). Soil nutrients = the total phosphorous (mg P/kg soil) of the soil microbial inoculum a seedling was grown in. Initial biomass = a seedling’s initial dry biomass (estimated). Observer = which of two researchers quantified AMF colonization in the seedling.

ANCOVA df F p

Soil nutrients 1, 5.32 0.25 0.27

Initial Biomass 1, 100.61 85.62 < 0.001 ***

Observer 1, 105.47 185.34 < 0.001 ***

Discussion

Our experiment revealed that soil microbes can vary in their impact on seedlings within the same plant population in a way that could promote genetic diversity within wild plant populations. Soil microbes near parent plants in our study population had a more negative impact on the growth of offspring seedlings relative to non-offspring seedlings (which are likely to be more distantly related), consistent with our hypothesis.

This growth difference was not driven by differences in soil nutrients or colonization by

AM fungi in the seedlings, suggesting that highly specialized soil pathogens were the primary mechanism. Our study provides the first evidence that soil microbes can produce a Janzen-Connell effect within a wild plant population. Such intraspecific Janzen-Connell effects could be important in maintaining genetic diversity within and among wild plant populations (Liu et al. 2015b, Browne & Karubian 2016). At the same time, our results suggest levels of genetic diversity within a plant population would influence plant- pathogen interactions and Janzen-Connell effects. For example, a recent study by Marden et al. (2017) of six tree species at our field site in Panama found that species having seedling cohorts with higher diversity of pathogen resistance genes suffered less from plant-soil feedbacks and negative density dependence compared to species having seedlings cohorts with lower resistance gene diversity. Their results, in combination with the results presented here, suggest that interactions between plants and genotype-specific pathogens may influence eco-evolutionary dynamics at both the population and community level.

Our plant-soil feedback analysis revealed a weak, but non-significant, trend towards negative plant-soil feedbacks within the population. Though negative plant-soil feedbacks are typically examined as a species-level phenomenon, the plant-soil feedback framework could apply within a given species if genotype-specific pathogens accumulate near conspecifics with genotypes they can infect, resulting in differences in soil microbial communities among conspecifics that reduce the survival of seedlings of host genotypes

(e.g., offspring) relative to seedlings of non-host genotypes. However, like at the species level, strict genotype-specificity of pathogens is probably not necessary for negative plant-soil feedbacks to maintain diversity (Sedio & Ostling 2013). Pathogen impacts must simply vary between two host genotypes, or, in the case of our study, between seedlings depending on their relatedness to the adult conspecific that cultured the soil microbial community. Plant-soil feedbacks could enhance local diversity within species as long as soil microbes have stronger negative effects on closely-related seedlings than on other conspecifics. Additional studies are needed to test for intraspecific plant-soil feedbacks in other plant species (ideally with more than 5 seed/soil sources) and in field conditions.

Our results suggest complex interactions between the effects of host-specific pathogens and arbuscular mycorrhizal fungi on seedling growth in plant populations.

Non-offspring seedlings in our experiment experienced higher colonization by AM fungi relative to offspring seedlings; however, differences in AM fungi colonization did not drive differences in the growth of the seedlings. Soil nutrients also did not drive colonization by AM fungi in the seedlings. Because pathogens and AM fungi sometimes compete within plant roots (Newsham et al. 1995), lower colonization by AM fungi in

offspring seedlings may have resulted from higher pathogen abundance in roots, rather than specialization of the AM fungi on seedlings that are more distantly related to their conspecific neighbors. Although colonization by AM fungi did not impact growth in our shadehouse experiment, decreased levels of colonization by AM fungi could have significant negative impacts on seedling growth in the long term under field conditions, i.e. where nutrients or water are more limited than the shadehouse. It is also important to note that the composition or relatedness of AM fungi within roots (which we did not identify in the present study) may play a larger role in determining the effectiveness of the mutualism than percent AM fungi colonization (Roger et al. 2013).

The same rapid co-evolutionary dynamics between plants and microbes that drive microbial host-specificity in agricultural and model plant species could have important ecological and evolutionary consequences in wild plant systems (reviewed by Stahl &

Bishop 2000, van der Does & Rep 2007, Whitham et al. 2012). While our study suggests that soil microbes can specialize on genotypes within single wild plant populations, further studies that explicitly test microbial genotype-specificity via cross-inoculation of microbial strains on plants of known genotypes will confirm genotype-specificity and reveal how often genotype-specificity occurs in wild plant-microbe systems. Our study also suggests that the genetic relationship between seedlings and neighboring adult conspecifics predicts seedling performance. This idea is supported by a study of a tropical palm population in Ecuador that found that the environments near conspecific adults in the field (including both soil microbes and herbivores) promoted the survival of seedlings with rare genotypes (Browne & Karubian 2016), but additional studies are needed that

explicitly measure genetic relatedness and test whether genetic relatedness (beyond offspring vs non-offspring) underlie differences in seedling performance near conspecifics. Our study suggests that the trend found by Browne & Karubian (2016) could have been driven by highly specialized soil microbes that accumulate near host plants, but additional field studies are needed (e.g. Mangan et al. 2010a) to confirm whether soil microbes drive dynamics within plant populations under natural environmental conditions. Our results further suggest that distinct soil microbial communities accumulate near plants in wild plant populations. Such differences in soil microbial communities could arise because of host control by plants (i.e., plant-soil feedback) or abiotic variation in soil environments (Schappe et al. 2017), and have been documented in a variety of plant species (reviewed by Whitham et al. 2012), suggesting widespread potential for highly specialized soil microbes to promote diversity in wild plants.

Our results also provide insight in to the role of seed dispersal as an enemy escape mechanism (Howe & Smallwood 1982). Seed dispersal is thought to help reduce fitness losses due to species-specific enemies that accumulate near the parent plant. Though it is known that seeds and seedlings dispersed to heterospecific sites often survive better than those in conspecific sites (reviewed by Comita et al. 2014), our study suggests that parent plants obtain fitness benefits even if their seeds disperse near another conspecific adult.

Increased performance of seedlings near non-parent conspecifics could be especially important for common plant species, which are likely to encounter a conspecific during

seed dispersal, or for species dispersed by animals, which are likely to seek out other fruiting conspecifics while foraging.

Our findings suggest an important role for specialist soil microbes in maintaining genetic diversity within wild plant populations and communities. Genetic diversity is crucial in allowing wild plant populations to persist through population bottlenecks, colonize new sites, and adapt to rapidly-evolving pathogens. Our results suggest that plant-pathogen eco-evolutionary dynamics operate like a feedback loop in which highly- specialized, genotype-specific pathogens select for genetic diversity in the resistance of their host plants; this diversity could then drive further pathogen adaptation (Marden et al. 2017). In addition, studies of the Janzen-Connell hypothesis and plant-soil feedbacks rarely assess variation in performance among seedlings near conspecifics, but our results suggest that density-dependent and distance-dependent seedling recruitment near conspecifics is probably more variable than typically assumed. Traditional species-level approaches to understanding pathogen impacts could obscure intraspecific variation that is important in structuring diversity and abundance in plant populations and communities.

Understanding the widespread patterns of plant diversity produced by specialist microbes is challenging due to the complexity of wild plant-microbe systems, but is important for our understanding of how diversity is maintained in wild plant populations and communities (Gilbert 2002, Alexander 2010).

Chapter 2: Tropical Tree Seedling Survival and Growth Near Mother

and Other Conspecific Adults in the Field

Abstract

The Janzen-Connell hypothesis describes how species-specific natural enemies promote species diversity in plant communities by limiting the survival of conspecific seedlings near adult plants. Recent experimental studies have provided the first evidence that genotype-specific natural enemies could cause variation in conspecific seedling performance near adults within species. Genotype-specific natural enemies could maintain genetic diversity within plant species by reducing the survival or growth of seedlings near genetically similar conspecific adults, or of seedlings with more common genotypes. We conducted a field experiment in Panama with four tropical tree species to test for variation in seedling performance between offspring seedlings and non-offspring seedlings near conspecific adults in their populations. We planted seedlings in the field near either their own mother tree or less-related conspecific tree and hypothesized that genotype-specific natural enemies would limit the growth and survival of seedlings planted near their own mother tree relative to seedlings planted near a conspecific tree to which they were less related. After eight months, we found that seedling survival and biomass were similar for seedlings planted near their own mother tree and seedlings

planted near a non-mother conspecific tree. This suggests that genotype-specific natural enemies, if present, do not have a strong impact on the performance of seedlings growing near conspecific adults in these species over that period (the first nine months after germination). Our findings suggest that first-year conspecific seedling recruitment near adults in tropical trees is determined by factors other than the genetic relationship between the seedlings and the adults.

Introduction

The Janzen-Connell hypothesis highlights the importance of species-specific natural enemies in contributing to the maintenance of species coexistence in highly diverse tropical tree communities by reducing the survival of conspecific seedlings near reproductive adults or when conspecific densities are high, (Janzen 1970, Connell 1971).

Subsequent theoretical studies have shown that conspecific seedling mortality due to species-specific natural enemies can enhance local species richness (Adler & Muller-

Landau 2005) and structure the relative abundance of species in tropical tree communities

(Mangan et al. 2010). In addition, experimental studies have provided evidence that patterns of conspecific distance- and density-dependent seedling mortality occur not just in tropical forests, but in a variety of plant communities worldwide, including temperate forests and grasslands (reviewed by Carson & Schnitzer 2008, Comita et al. 2014).

The impact of natural enemies on tropical tree diversity has been examined primarily as a species-level effect, in which natural enemy species reduce the survival of conspecific seedlings relative to heterospecific seedlings near their host species. Within

the past decade, experimental studies have provided evidence for Janzen-Connell effects at additional ecological levels, such as phylogenetic Janzen-Connell effects due to natural enemies that specialize more broadly on closely related species (Bagchi et al. 2010, Liu et al. 2012), and intraspecific Janzen-Connell effects due to natural enemies that specialize more narrowly on genotypes within species (Liu et al. 2015, Browne & Karubian 2016).

In a shadehouse experiment, Liu et al. (2015) found that genetic relatedness between conspecific seedlings and adults predicted seedling survival in the soil microbial communities near those adults in two subtropical tree species in China: soil microbial communities promoted the survival of seedlings from more genetically distant populations. Such studies provide evidence suggesting that seedling survival near adult trees is determined not just by species-specific impacts of natural enemies, but by the degree of phylogenetic or genetic similarity between seedlings and adults. A theoretical study showed that distance-respondent natural enemies do not need to be strictly species- specific to maintain tree species coexistence - they simply need to affect each of their host species differently (Sedio & Ostling 2013). Another theoretical study showed that intraspecific variation in the suitability of conspecific adult sites for seedling recruitment can contribute to diversity even in the absence of conspecific distance-dependent seedling mortality (Stump & Chesson 2015). Like traditional Janzen-Connell effects, phylogenetic and intraspecific distance- and density-dependent seedling mortality grant an advantage to seedlings that are different from established trees and create variation in seedling recruitment among sites. These additional Janzen-Connell effects could also be important

in structuring and maintaining tree diversity, specifically by promoting phylogenetic diversity within tree communities or genetic diversity within tree species or populations.

Though intraspecific Janzen-Connell effects are beginning to be explored, few studies have examined their potential occurrence among adult conspecifics within a population. In a prior shadehouse experiment with the tropical tree Virola surinamensis

(Myristicaceae) in Panama (Chapter 1), we found evidence of an intraspecific distance- dependent effect on seedling growth resulting from the soil microbial communities near adult conspecifics within a single tree population. Specifically, we found that the soil microbial communities of conspecific adults increased the biomass of non-offspring seedlings relative to offspring seedlings, suggesting a recruitment advantage for seedlings that are more genetically distant from the adult they are near. In addition, a recent experiment with the tropical palm Oenocarpus bataua in Ecuador showed that seedlings with rarer genotypes (at the population level) were more likely to survive in the field near conspecific adults than seedlings with more common genotypes, providing evidence for intraspecific density-dependent survival of seedling genotypes within a population

(Browne & Karubian 2016). To date, no existing studies have tested for within- population intraspecific distance-dependent seedling mortality in the field where seedlings are exposed to their full set of natural enemies.

We conducted a field experiment in Panama with four tropical tree species to search for new evidence of intraspecific distance-dependent seedling survival near adult conspecifics within tropical tree populations. Our experiment tested the hypothesis that genotype-specific natural enemies reduce the survival and biomass of offspring seedlings

relative to non-offspring conspecific seedlings growing near adult trees. By examining the conspecific seedling-adult relationships as a potential source of intraspecific variation in seedling survival near adult conspecifics, we hope to gain a better understanding of how intraspecific Janzen-Connell effects might impact diversity within tropical tree populations and communities.

Methods

Study site and species

We conducted our study on Barro Colorado Island (BCI), Republic of Panama

(9°09’ N, 79°51’ W). BCI is a 15.6 km2 lowland, tropical moist forest (Croat 1978). BCI is seasonally dry, receiving an average of 2623 mm of rain per year, punctuated by a distinct dry season (Windsor 1990). BCI was isolated from the surrounding mainland by the creation of Gatun Lake in 1914, during the building of the Panama Canal.

We chose four medium or large canopy tree species on BCI as focal species for the study: Lacmellea panamensis (Woodson) Markgf. (Apocynaceae), Ormosia macrocalyx Ducke (Fabaceae), Tetragastris panamensis (Engl.) Kuntze (Burseraceae), and Virola surinamensis (Rol. ex Rottb.) Warb. (Myristicaceae). Virola surinamensis, T. panamensis, and O. macrocalyx are native to much of Central America and the northern

Amazon, while L. panamensis is limited to Panama, Costa Rica, and Belize (Croat 1978).

The species vary in abundance on BCI: V. surinamensis and T. panamensis are common,

L. panamensis is occasional, and O. macrocalyx is rare (Croat 1978). These species were chosen because seedlings of all the species are shade-tolerant (Howe 1990, Gilbert et al.

2006, Myers & Kitajima 2007, Krause et al. 2012). Seedlings of T. panamensis and L. panamensis are drought-tolerant, while seedlings of V. surinamensis are drought- sensitive (Kursar et al. 2009) (information on the drought tolerance of O. macrocalyx seedlings could not be found). On BCI, V. surinamensis is associated with slope habitats,

T. panamensis with low plateau habitats, and L. panamensis with swamp habitats (Harms et al. 2001) (information on the habitat preference of O. macrocalyx could not be found).

Flowering occurs in the dry season for V. surinamensis, in the wet season for T. panamensis and O. macrocalyx, and from the late dry season to early wet season for L. panamensis. Seed rain peaks in March for T. panamensis, April for L. panamensis, June for V. surinamensis, and in the dry season (month unspecified) for O. macrocalyx (Croat

1978, Zimmerman et al. 2007). Seeds of each species are medium or large and animal- dispersed: V. surinamensis (~2 cm), T. panamensis (~1 cm) and L. panamensis (~1 cm) seeds are enclosed in nutritious fruits or arils that attract large birds or monkeys as dispersers (Howe 1980, Howe & Vande Kerckhove 1981, Wehncke & Dalling 2005). O. macrocalyx seeds (~1 cm) are hard and yield no nutritious reward, but occasionally attract bird dispersers mimetically (Foster & Delay 1998). V. surinamensis and T. panamensis are dioecious (seeds and pollen are produced on separate individuals), while

L. panamensis and O. macrocalyx are hermaphroditic (seeds and pollen are produced on the same individual).

Field experiment

We collected ~3600 seeds from the ground beneath the canopy of 23 fruiting V. surinamensis, ~750 seeds from beneath 11 fruiting T. panamensis, ~650 seeds from beneath 12 fruiting L. panamensis, and ~600 seeds from beneath 3 O. macrocalyx on BCI in June and July 2015. We assigned the tree that a seed was collected beneath as the seed’s putative mother, and kept seeds from each maternal source separate. Trees were located by exploring a ~3.5 km2 area and with the aid of a mapped 25 ha plot (provided by Wright, S. J., personal communication). The average number of seeds collected per tree was 156 for V. surinamensis, 69 for T. panamensis, 60 for L. panamensis, and 186 for O. macrocalyx. After collection, all seeds were surface sterilized (10% bleach for 2 min., rinse, 70% ethanol for 2 min., rinse), air dried, planted in germination trays

(containing autoclaved BCI soil) in the shadehouse, and kept well-watered under two layers of 80% shadecloth. O. macrocalyx seeds were submerged in water for 24 hours prior to planting to encourage germination (Sautu et al. 2006). Seed germination rates varied by species: ~7% in V. surinamensis and T. panamensis, ~48% in O. macrocalyx, and ~22% in L. panamensis. Six V. surinamensis seed sources, three T. panamensis seed sources, seven L. panamensis seed sources, and three O. macrocalyx seed sources provided sufficient numbers of seedlings healthy in appearance one month after germination for inclusion in the experiment. These 19 maternal seed sources were chosen as focal trees in the study, and provided a total of 373 experimental seedlings for use in the experiment (145 V. surinamensis, 130 O. macrocalyx, 68 L. panamensis, and 30 T. panamensis). A few additional reproductive-size trees (beyond the 19 maternal seed

sources) from within the study area on BCI were also included as focal trees in the experiment (to serve as additional transplant sites) (one female T. panamensis, and two

O. macrocalyx). Distances between pairs of focal trees range from ~30 m to ~2 km.

Within each focal species, experimental seedlings were assigned to one of two experimental field treatment groups: ‘mother environment’ or ‘other conspecific environment’. Seedlings in the mother group were assigned for transplant beneath the canopy of their own (putative) mother tree; seedlings in the other conspecific group were assigned for transplant beneath the canopy of one of the conspecific focal trees in their species’ experiment that was not their own mother. For seedlings in the other conspecific group, which non-mother conspecific tree each seedling was assigned to was chosen at random. Sample sizes for each species, treatment, and combination of maternal seed source and focal adult transplant site in the experiment can be found in Table 9.

Table 9: Sample sizes for each species and treatment in the ‘mother vs. other’ field experiment. Panels A through D show sample sizes for treatments and combinations of maternal seed source and focal adult (transplant site) for each species’ experiments (Panel A: Lacmellea panamensis; Panel B: Ormosia macrocalyx; Panel C: Virola surinamensis; Panel D: Tetragastris panamensis). Panel E shows overall sample sizes for the entire experiment when all species are pooled.

Continued

Table 9: Continued

Seedlings were randomly assigned to one of several 1 m2 seedling plots beneath the canopy of their assigned tree. Plot placement beneath a tree’s canopy was randomized with respect to direction and distance from the base of the tree (1-4 m). Focal species varied in the average number of seedling plots beneath their focal trees, and in the average number of seedlings in each plot (species with more experimental seedlings had more plots per focal tree and more seedlings per plot). Within a focal species, focal trees that provided more experimental seedlings (i.e., maternal trees providing more seedlings relative to those providing less seedlings, or maternal trees relative to non-seed source conspecific focal trees) had more plots beneath their canopies to accommodate their offspring seedlings without increasing the number of seedlings per plot. Focal trees had between one and seven plots beneath their canopies. Each plot contained between two and five experimental seedlings, depending on the species: two or three for T. panamensis, four or five for V. surinamensis, four or five for O. macrocalyx, and three, four, or five for L. panamensis. The relatively small range in number of seedlings per plot

(hereafter referred to as “conspecific seedling density”) was chosen to minimize the potential impact of conspecific seedling neighbors on seedling growth or survival (i.e., negative density-dependent mortality) and was not intended to be a variable of focus in the study. In the O. macrocalyx experiment, seedlings in the mother treatment were planted at higher conspecific seedling densities than seedlings in the other conspecific treatment (conspecific seedling densities were similar between the two treatments for the other species’ experiments). Seedlings in each plot were randomly assigned to one of

sixteen 25 cm2 positions within the plot (if the chosen position was already occupied by a plant or a rock, a different position was chosen at random).

All experimental seedlings were transplanted into their experimental plots in the field between August and October 2015, at ~1 month of age. Focal species were transplanted one at a time. At the time of transplant, seedlings were tagged with a unique identification number and initial stem height, leaf number, and leaf area (length and width) were measured (see Figure 12 for a photograph of a tagged experimental seedling). Seedlings were censused every 1-2 months, during which their status

(alive/dead) was recorded and stem height and leaf number measured (for the surviving seedlings). Initial dry biomass was estimated for each experimental seedling based on height and estimated leaf area (based on measurements of leaf length and width) at the time of transplant using species-specific allometric linear regression models (V.

2 - surinamensis: F2,47 = 496.5, p < 0.001, R = 0.95; O. macrocalyx: F2,37 = 154.7, p < 2.2e

16 2 -16 2 , R = 0.89; L. panamensis: F2,26 = 183.7, p = 4.6e , R = 0.93; T. panamensis: F2,7 =

19.7, p = 0.001, R2 = 0.81). These allometric models were created using measurements of the height, leaf area (measured with a leaf area machine), and dry biomass of a randomly harvested sample of the potential experimental seedlings of each species.

Figure 12: A photograph of a tagged experimental seedling in a field plot.

We also recorded the status of physical damage to the seedlings during each census, such as stem breakage (likely by agoutis or other mammalian herbivores), as

‘clipping’ (clipped/unclipped). Clipping was common in the V. surinamensis experiment

– clipping was observed at some point during the experiment in 46 out of the 145 V. surinamensis seedlings (31.7%). Clipping was rarer in the other species (6.6% in T. panamensis, 4.6% in O. macrocalyx, and 16.2% in L. panamensis). In addition, some seedlings were uprooted between censuses (perhaps by peccaries, or by torrential rain), and were found lying nearby. Uprooting was occasional in V. surinamensis (14 out of 145 experimental seedlings, 9.7%) and L. panamensis (5 out of 68 experimental seedlings,

7.4%). Some seedlings decayed, were destroyed, or otherwise disappeared between

censuses, leaving no remnants of the dead seedling that could be found (the tags usually were found for these seedlings). The clipping and/or uprooting status of these disappearing seedlings could not be determined, meaning that actual clipping or uprooting rates could be higher than we observed.

After ~7-8 months in their field treatments, all surviving seedlings were harvested and brought to the lab, where their total oven-dried biomass, stem height, and leaf area were measured (leaf area was measured with a leaf area machine). Seedlings were harvested one species at a time between March and May 2016. The final months of the experiment overlapped with a severe dry season due to the strong 2015/16 El Niño event; signs of wilting were observed in some seedlings, particularly V. surinamensis.

Statistical analyses

We analyzed the effect of the experimental field treatment (i.e., planting beneath the canopy of a seedling’s own mother tree or a non-mother conspecific tree) on seedling survival at harvest using a generalized linear mixed-effects model with binomial distributions. Due to small sample sizes in some focal species, seedlings from all species were pooled for survival analysis, and species was included as a fixed effect in the model.

The number of experimental seedlings in the seedling’s plot (i.e., conspecific seedling density) was included as a fixed effect in the model. To account for other potential sources of variation in seedling survival, we included maternal seed source, focal adult tree (i.e., transplant site), and plot as random effects in the model. To account for the potential impact of variation in the size of the seedlings at the beginning of the

experiment, we also included an estimate of initial seedling dry biomass as a covariate in the model. We also included clipping during the experiment (clipped/unclipped) as a binary fixed effect in the model. Uprooted seedlings were not included in the model.

We also analyzed the effect of the experimental field treatment on seedling dry biomass at harvest using a linear mixed-effects model. As above, seedlings from all species were pooled for the biomass analysis, and species was included as a fixed effect in the model. Dead and/or uprooted seedlings were not included in the model. As above, conspecific seedling density, an estimate of initial seedling biomass, and clipping were included as fixed effects in the model, and maternal seed source, focal adult tree, and plot were included as random effects.

The mixed-effects analyses in our study were performed using the lme4 package

(Bates et al. 2015) in the R environment (R Core Team 2017). R2 values for the mixed- effects analyses were obtained with the MuMIn package (Barton 2016). P-values for mixed-model predictors were obtained with the lmerTest package (Kuznetsova et al.

2016). ANOVA tables for the mixed-effects models were obtained with the afex package

(Singmann et al. 2017).

Results

Seedling survival in mother vs. other conspecific environments

The overall survival rate in the field experiment was 35.6% (126 out of 354 seedlings), while the survival rate in the mother treatment group was 31.7% (48 out of

154 seedlings) and the survival rate in the other conspecific treatment group was 39% (78

out of 200 seedlings) (Table 10). Seedlings of O. macrocalyx were significantly more likely to survive than seedlings of any other species. Our generalized linear-mixed effects model revealed that the probability of seedling survival was nearly identical for seedlings that were planted near their own mother and seedlings that were planted near a non- mother conspecific (Table 11, Figure 13). Mean fitted seedling survival probabilities were higher in other conspecific environments relative to mother environments in 10 out of the 19 maternal seed sources in the experiment (Figure 14). Mean fitted seedling survival probabilities were also higher for non-offspring seedlings relative to offspring seedlings near 9 out of the 17 focal adults for which data to compare these two groups was available (Figure 15). Seedlings planted at different conspecific seedling densities had similar survival probabilities, with a non-significant trend towards higher survival probabilities at intermediate conspecific seedling densities (Figure 16). Seedlings that were larger at the beginning of the experiment (i.e., those with higher initial biomass) had a significantly higher probability of survival. Clipping did not have a strong effect on seedling survival, with a non-significant trend towards lower survival probabilities for clipped seedlings.

Table 10: Survival of seedlings in the ‘mother vs. other’ field experiment.

Table 11: Mixed-model ANCOVA of seedling survival in mother vs. other conspecific environments in the field. Panel A: ANCOVA table of the predictors of seedling survival at the end of the ~8-month field experiment (n = 354 seedlings) including degrees of freedom (df), F-score (F), and p-value (p). Panel B: Fixed effects summary includes logit-linked coefficient estimates (β), standard errors (SE(β)), z-score (z = β/SE(β)), and significance level (p) of each fixed effect in the model. Intercept = the probability of survival of unclipped L. panamensis seedlings in mother environments; other conspecific environment = the impact of the non-mother conspecific environment on survival; conspecific seedling density = the impact of adding one conspecific seedling to a plot on seedling survival; clipping = the impact of being clipped on seedling survival; initial biomass = the impact of initial biomass on seedling survival; species = the impact of being another species on seedling survival. Panel C: Random effects summary includes variance and standard deviation of each random effect in the model (n = 19 maternal seed sources, 22 focal adults, and 90 seedling plots). Mixed-model R2 = 0.64.

Panel A: ANCOVA df F p Conspecific Environment 291.91 0.00 0.96 Conspecific Seedling Density 84.89 2.77 0.10 Initial Biomass 138.07 23.79 <0.0001 Clipping 307.76 2.57 0.11 Species 20.83 9.53 0.004

Panel B: Fixed Effects β SE(β) z p Intercept 0.50 2.03 0.25 0.81 Other Conspecific Environment -0.01 0.36 -0.03 0.98 Conspecific Seedling Density -0.84 0.52 -1.61 0.11 Initial Biomass 8.41 1.95 4.32 1.56e-05 Clipping -0.87 0.59 -1.48 0.14 Species: O. macrocalyx 2.69 0.99 2.70 6.84e-03 Species: T. panamensis -0.82 1.19 -0.68 0.49 Species: V. surinamensis -1.50 1.17 -1.28 0.20

Panel C: Random Effects Var. SD Maternal Seed Source 0.00 0.00 Focal Adult 1.06 1.03 Plot 1.82 1.35

Figure 13: Seedling survival in mother vs. other conspecific environments in the field. Fitted survival probabilities for seedlings planted near their own mother vs. seedlings planted near a different conspecific adult at the end of the ~8-month field transplant experiment (n = 354 seedlings: 154 in the mother group and 200 in the other conspecific group). A generalized linear mixed-effects model revealed that there was not a significant effect of conspecific environment on seedling survival (p = 0.98). Whisker lengths are + 1.5*IQR.

Figure 14: Survival of seedlings from 19 maternal seed sources in mother and other conspecific environments the field. The fitted probability of survival of seedlings from 19 maternal seed sources in mother vs. other conspecific environments in the field (n = 354 seedlings). Mean survival probabilities were higher in other conspecific environments relative to mother environments in 10 out of the 19 maternal seed sources. Whisker lengths are + 1.5*IQR.

Figure 15: Survival of offspring vs. non-offspring conspecific seedlings near 22 focal adults in the field. The fitted probability of survival of offspring vs. non-offspring seedlings near 22 focal adults in the field (n = 354 seedlings). NAs represent missing treatment combinations. Fitted survival probabilities were higher for non-offspring seedlings relative to offspring seedlings near 9 out of the 17 focal adults for which comparisons of these two groups was possible. Whisker lengths are + 1.5*IQR.

Figure 16: Seedling survival across conspecific seedling densities in the ‘mother vs. other’ field experiment. Fitted survival probabilities for seedlings planted at conspecific densities = 2, 3, 4 or 5 at the end of the ~8-month field transplant experiment (n = 354 seedlings). A generalized linear mixed-effects model revealed that there was a marginally-significant effect of conspecific seedling density on seedling survival (p = 0.10).

A linear mixed-effects model revealed that biomass was similar for seedlings that survived near their own mother and seedlings that survived near a non-mother conspecific, with a non-significant trend towards lower biomass for seedlings planted near a non-mother conspecific (Table 12, Figure 17). Biomass was higher in other conspecific environments relative to mother environments in seven out of the 11 maternal seed sources where biomass data were available for both groups (Figure 18) (missing data due to dead seedlings made comparisons impossible for some seed sources and adult sites). Biomass was also higher for non-offspring seedlings relative to offspring seedlings near five out of the nine focal adults where biomass data were available for both groups

(Figure 19). Seedlings planted at different conspecific seedling densities also had similar biomass, with a marginally-significant trend towards lower biomass at higher conspecific seedling densities (Figure 20). The seedlings did not grow much overall: mean initial biomass was estimated at 0.19 g, while mean final biomass was 0.32 g. Seedlings that were larger at the beginning of the experiment (i.e., those with higher initial biomass) had significantly higher final biomass. Clipping also had a strong effect on biomass: clipped seedlings had significantly lower final biomass. Seedlings of V. surinamensis had significantly higher biomass than seedlings of the other species.

Table 12: Mixed-model ANCOVA of seedling biomass in mother vs. other conspecific environments in the field. Panel A: ANCOVA table of the predictors of seedling biomass at the end of the ~8-month field experiment (n = 126 seedlings) including degrees of freedom (df), F-score (F), and p-value (p). Panel B: Fixed effects summary includes coefficient estimates (β), standard errors (SE(β)), t-score (t = β/SE(β)), and significance level (p) of each fixed effect in the model. Intercept = the biomass of unclipped L. panamensis seedlings in mother environments; other conspecific environment = the impact of being in a non-mother conspecific environment on biomass; conspecific seedling density = the impact of adding one conspecific seedling to a plot on biomass; clipping = the impact of being clipped on biomass; initial biomass = the impact of initial biomass on final biomass; species = the impact of being another species on biomass. Panel C: Random effects summary includes variance and standard deviation of each random effect in the model (n = 17 maternal seed sources, 18 focal adults, and 55 seedling plots). Mixed-model R2 = 0.78.

Panel A: ANCOVA df F p Conspecific Environment 112.24 2.18 0.14 Conspecific Seedling Density 48.36 1.02 0.32 Initial Biomass 108.44 64.99 1.08e-12 Clipping 109.58 16.63 8.64e-05 Species 18.67 5.46 7.21e-03

Panel B: Fixed Effects β SE(β) t p Intercept 0.17 0.08 2.11 0.04 Other Conspecific Environment -0.03 0.02 -1.48 0.14 Conspecific Seedling Density -0.02 0.02 -1.01 0.32 Initial Biomass 0.68 0.08 8.06 1.08e-12 Clipping -0.15 0.04 -4.08 8.64e-05 Species: O. macrocalyx 0.09 0.05 1.79 0.09 Species: T. panamensis 0.04 0.06 0.67 0.51 Species: V. surinamensis 0.24 0.06 3.95 6.39e-04

Panel C: Random Effects Var. SD Maternal Seed Source 2.09e-05 0.08 Focal Adult 4.21e-03 0.06 Plot 2.21e-04 0.01 Residual 6.52e-03 0.08

Figure 17: Seedling biomass in mother vs. other conspecific environments in the field. Biomass of seedlings planted near their own mother vs. seedlings planted near a different conspecific adult at the end of the ~8-month field transplant experiment (n = 126 seedlings: 48 in the mother group and 78 in the other conspecific group). A linear mixed-effects model revealed that there was not a significant effect of conspecific environment on seedling biomass (p = 0.14). Whisker lengths are + 1.5*IQR.

Figure 18: Biomass of seedlings from 19 maternal seed sources in mother and other conspecific environments the field. The biomass of seedlings from 19 maternal seed sources in mother vs. other conspecific environments in the field (n = 126 seedlings). NAs represent missing values due to dead seedlings or non-existent treatment combinations. Mean biomass was higher in other conspecific environments relative to mother environments in 7 out of the 11 maternal seed sources where biomass data were available for both groups. Whisker lengths are + 1.5*IQR.

Figure 19: Biomass of offspring vs. non-offspring conspecific seedlings near 22 focal adults in the field. The biomass of offspring vs. non-offspring seedlings near 22 focal adults in the field (n = 126 seedlings). NAs represent missing values due to dead seedlings or non-existent treatment combinations. Mean biomass was higher for non- offspring seedlings relative to offspring seedlings near 5 out of the 9 focal adults for which comparisons of these two groups was possible. Whisker lengths are + 1.5*IQR.

Figure 20: Seedling biomass across conspecific seedling densities in the ‘mother vs. other’ field experiment. Fitted biomass for seedlings planted at each conspecific seedling density at the end of the ~8-month field transplant experiment (n = 126 seedlings). A linear mixed-effects model revealed that there was not a significant effect of conspecific seedling density on seedling survival (p = 0.32).

Discussion

In our field experiment, we did not find evidence that the relationship between seedlings and the conspecific adult they are growing near has a strong influence on tropical tree seedling survival or growth. Seedlings that were growing near their own mother tree (a close relative) survived and grew the same as seedlings that were growing near a different (presumably less-related) adult in the population. Variation in seedling survival or growth near conspecific adults could result from genotype-specific natural enemies that limit the performance of seedlings that share defensive characteristics with established plants relative to seedlings that are more distantly related. Recent experiments in the field (Browne & Karubian 2016) and greenhouse (Chapter 1) have given the first empirical evidence of the patterns of survival and growth predicted by genotype-specific natural enemies, indicating that such enemies could be important in determining dynamics in plant populations and communities. Though we did not find evidence of the patterns of survival or growth that would be expected if natural enemies are genotype- specific in our field study, genotype-specific natural enemies could still play an important role in determining patterns of diversity and abundance in plant populations and communities.

We also did not find evidence indicating that conspecific seedling density plays an important role in mediating seedling performance near conspecifics. The seedlings in our study showed a general trend towards reduced survival and growth with increases in conspecific seedling density, but these trends were not strong enough to be statistically

significant. Patterns of negative density-dependence due to conspecific seedling neighbors are common in tropical trees and are often the result of the actions of natural enemies (reviews of negative density-dependence can be found in Carson & Schnitzer

2008, Comita et al. 2014). A long-term observational study of seedling survival on BCI found evidence that density-dependent seedling mortality due to conspecific adults and seedling neighbors is relatively common among tree species at our field site (Comita et al. 2010). Given the relatively small range of conspecific seedling densities utilized in our study (i.e., one additional conspecific seedling per square meter plot), the lack of a significant impact of conspecific seedling density on seedling performance in our study is not surprising. In fact, the small range of variation in conspecific seedling density chosen among the plots in our study was intended to minimize negative impacts of density dependence due to conspecific neighbors. The fact that any negative trend in survival and growth could be detected over such small variation in conspecific seedling density suggests that natural enemies respond even to small variation in conspecific seedling density, and that ultimately, this response has an impact on seedling performance.

Conspecific seedling neighbors are known to be important in contributing to density- dependent seedling mortality (Queenborough et al. 2007, Comita et al. 2010, Metz et al.

2010, Lebrija-Trejos et al. 2014), but it is not well-understood how conspecific seedling neighbors might contribute to genotype-specific impacts of natural enemies. Conspecific seedling neighbors reduce seedling survival by competing for shared resources or by increasing natural enemy densities. Our previous shadehouse experiment showed that, in isolation from conspecific neighbors, offspring seedlings that are more related to nearby

adults are at a growth and mutualist disadvantage relative to non-offspring seedlings. The advantage of seedlings with a locally rare genotype (i.e., non-offspring seedlings near a parent tree) might strengthen in the presence of conspecific neighbors that increase the densities of natural enemies tuned to the locally common genotype (i.e., the genotype of the parent tree and its offspring). Though conspecific negative density-dependence mandates that the survival of both offspring and non-offspring seedlings is generally expected to decrease as conspecific seedling neighbor densities increase, offspring and non-offspring seedlings near adult conspecifics may not be equally impacted by intraspecific negative density-dependence if seedling genotype plays a role in determining the impacts of the local natural enemies.

This study adds to the findings of our prior greenhouse study by informing us about the impact of genotype-specific natural enemies. In a prior shadehouse experiment, we found evidence of variation in V. surinamensis seedling growth and AMF colonization when seedlings were grown singly in the soil microbial community of their mother relative to the soil microbial community of a non-mother female conspecific in the shadehouse (Chapter 1). In that study, soil microbes provided an advantage in terms of growth and association with mutualists to non-offspring seedlings in conspecific adult soils. In the present study, we did not find that biomass was reduced for offspring seedlings near their mother trees. In addition, we did not find that the survival or growth of non-offspring conspecific seedlings was increased relative to offspring seedlings overall near conspecific adults in the field. It is possible that growth advantages to non- offspring conspecific seedlings due to soil microbes do also occur in the field, but are not

strong enough to be observed amidst the other factors influencing seedling survival and growth. In a long-term field experiment, Browne & Karubian (2016) found that conspecific seedlings of a tropical palm species with rarer genotypes (at the population level) were more likely to survive near conspecific adults, a form of intraspecific density- dependent mortality of seedlings with common genotypes. Seedlings in the non-offspring treatment in our study likely represented different genotypes than the adult conspecifics they were near, but we did not find evidence of a corresponding negative impact of conspecific environments on offspring seedlings with locally common genotypes (i.e., offspring seedlings).

If genotype-specific natural enemies and/or mutualists do sometimes cause variation in seedling performance or survival near conspecifics in the field, there are several potential reasons why our study may not have detected such effects. Because our seedlings were already one month of age when the experiment began, our study could not possibly detect effects that might impact the survival of seeds or that impact seedling mortality very soon after germination. It is also possible that the duration of our study (~

8 months) was too short for strong effects on seedling mortality or growth to develop, but that such effects might develop over longer time periods. In addition, though there was a fair amount of seedling mortality in our study, the cause of death was not usually known, and several other environmental factors (besides natural enemies) could have been the primary drivers of seedling survival in our study. For example, wilting was observed during the dry season in some seedlings, presumably due to drought stress. In addition, low growth rates during the study period and low survival rates reduced the power of our

growth analysis. Clipping and uprooting, likely by mammalian herbivores, also affected many seedlings in the study (mainly V. surinamensis), reducing the power of our study to detect the effects of soil microbes, which have been previously shown to favor the growth of non-offspring seedlings of V. surinamensis in conspecific soils. Genetic analyses are also needed to confirm our experiment’s assumption of parentage of the mother trees to the seedlings assigned as putative offspring. It is possible that some of the offspring- mother pairs in our study are erroneous, if any seeds we collected from beneath the canopy of a mother had been deposited there previously by animal dispersers. Genetic analyses are also needed to examine whether genetic relatedness is reduced between non- offspring-mother pairs relative to offspring-mother pairs in our hermaphroditic focal species (L. panamensis and O. macrocalyx). In these species, it is possible that other conspecific trees could be pollen donors (i.e., fathers) to any experimental conspecific seedling in our study. In the dioecious species, the other conspecific trees were all females that could not donate pollen, ensuring that non-offspring-mother pairs are less genetically related than offspring-mother pairs.

We examined the impacts of the relationship between conspecific seedlings and adults and conspecific seedling neighbors on seedling survival and growth near adult conspecifics in several tropical tree species. Though we did not find evidence that genotype-specific natural enemies impact seedling performance in the field, it’s possible that such effects may be present in other species, at other sites, or under different environmental conditions. Intraspecific variation in seedling performance near conspecific adults could be important because of its potential to promote tropical tree

seedling diversity (particularly locally rare genotypes), and maintain the coexistence of genotypes within tropical tree populations. Beyond improving our understanding of population dynamics, understanding intraspecific Janzen-Connell effects will also help us to model the impact of natural enemies on species diversity in tree communities. If conspecific trees within species vary in their limitation of conspecific seedling recruitment, this creates variation among sites in the enhancement of local species richness. Further study of intraspecific Janzen-Connell effects should provide a better understanding of how distance- and density-dependent seedling mortality near adult and seedling conspecifics might impact diversity within tropical tree populations and communities.

Chapter 3: Conspecific Seedling Survival, Growth, and Colonization by

Arbuscular Mycorrhizal Fungi near Female and Male Adults in a

Dioecious Tropical Tree Species

Abstract

Natural enemies, such as pathogens and herbivores, play an important role in promoting species diversity in plant communities by limiting the recruitment of conspecific seedlings near adult plants. The input of conspecific seeds into the environment during seed production is an important factor contributing to natural enemy accumulation near adults, but the impact of variation in seed production on conspecific seedling recruitment has rarely been studied. Dioecious species offer a unique opportunity to study the impact of variation in seed production on conspecific seedling recruitment, because male plants never produce seeds, reducing seed input beneath their canopies. We hypothesized that seedling performance would be reduced under reproductive female trees relative to male trees due to differences in soil microbial communities arising from the higher seed input under females. To test this hypothesis, we conducted parallel shadehouse and field experiments to examine the impact of seed production on conspecific seedling recruitment near female and male adults of a dioecious tropical tree species, Virola surinamensis (Myristicaceae), in Panama. The

shadehouse experiment isolated the influence of male vs. female soil microbial communities on seedling growth, as well as on colonization of seedlings by arbuscular mycorrhizal fungi (AMF). We also quantified seed production by females, and examined whether variation in the amount of seed input influences seedling biomass or colonization by AMF among female soils. In the field experiment, we planted seedlings beneath the canopy of reproductive-size male and female V. surinamensis trees and monitored seedling survival and growth for eight months. We found that seedling growth and colonization by AMF were similar in male and female soil microbial communities in the shadehouse. This suggests that seed input into soils by females does not alter soil microbial communities in a way that impacts seedling performance in those soils. We also did not find a relationship between the amount of seed input and seedling growth or colonization by AMF in female soils. In the field, seedling survival and growth were also similar beneath male and female adults. Overall, our experiments show that seed input beneath female trees does not decrease conspecific seedling survival and growth near females relative to males in this species. Though it is possible that seed input could cause short- or long-term changes to the density or composition of natural enemy communities near fruiting plants, our experiments suggest that any such changes do not have a large influence on conspecific seedling recruitment during the first year. Significant effects of seed production on conspecific seedling recruitment could still emerge over longer time periods, during later developmental stages, or could be critically determined during or immediately after seed germination. The generality of our findings will be revealed by studies examining the impact of seed production on conspecific seedling recruitment in

additional plant species and communities. We suggest that future studies test for short- and long-term changes in natural enemy composition or density near plants due to seed production, and also examine the impact of other factors that could be important in contributing to the accumulation of natural enemies near plants, such as adult size, neighborhood effects, or abiotic effects. Understanding the biotic and abiotic factors that contribute to the accumulation of natural enemies and conspecific recruitment limitation near adult plants, as well as the sources and consequences of variation in these processes, will help us better understand how natural enemies structure plant diversity and abundance.

Introduction

The Janzen-Connell hypothesis describes how host-specific natural enemies, such as pathogens and herbivores, maintain species diversity in plant communities by accumulating near their host plants and limiting the recruitment of conspecific seedlings beneath their canopies (Janzen 1970, Connell 1971). Theoretical studies have demonstrated that the distance- and density-dependent patterns of recruitment predicted by the Janzen-Connell hypothesis can enhance local species richness (Adler & Muller-

Landau 2005) and structure the relative abundance of species in a community (Mangan et al. 2010). The demographic patterns predicted by Janzen (1970) and Connell (1971) have been shown to occur in a variety of plant communities worldwide (reviewed by Carson &

Schnitzer 2008, Comita et al. 2014). In fact, a recent study found that the strength of

negative conspecific density-dependence in a tree community is correlated with species richness of the community (LaManna et al. 2017).

Despite the importance of natural enemies in structuring plant diversity and abundance, it is not well-understood which factors of plants or their environments are important in determining the density of natural enemies that accumulate near them, and, ultimately, the strength of negative distance- and density-dependence experienced by conspecific seedlings that attempt to recruit near them. Within a species, host factors such as size, reproduction, or genotype could cause the density of natural enemies to vary near established plants. In addition, characteristics of the environment near plants, such as the composition of surrounding plants (especially other conspecifics) (Liang et al. 2016) or the abiotic conditions of a plant’s habitat (e.g., soil fertility or water or light availability), could also contribute to variation in natural enemy accumulation among plants within a species. Though much focus has been placed on showing patterns of negative conspecific distance- and density-dependence and determining whether such patterns are caused primarily by pathogens, herbivores, or competition with neighbors (Chanthorn et al.

2010, Kotanen 2010, Johnson et al. 2014, Lebrija-Trejos et al. 2014), we know less about how much this process varies among adults within species, or about the causes and consequences of such variation.

Seed production is one factor that likely has an impact on natural enemy densities and conspecific seedling recruitment near adult plants. Because of limited seed dispersal distances, many conspecific seeds fall beneath or close to parent trees. Conspecific seeds and seedlings have been shown to contribute to density-dependent mortality in tropical

tree seedlings (Harms et al. 2000, Wright et al. 2005, Queenborough et al. 2007, Comita et al. 2010, Metz et al. 2010, Lebrija-Trejos et al. 2014), either by increasing local host- specific natural enemy densities or by increasing intraspecific competition for shared seedling resources. In this way, variation in seed production among adult plants within species could cause the recruitment success of nearby conspecific seedlings to vary: conspecific seedling mortality might be increased near plants that produce more seeds relative to those that produce fewer seeds. This hypothesis has rarely been tested (but see

Hood et al. 2004), despite the potential for variation in conspecific seedling survival near adults to create variation in how strongly natural enemies enhance local species richness.

If seed production has direct or indirect impacts on the recruitment success of conspecific seedlings, the impact of variation in seed production would likely be most pronounced near adults of dioecious plant species, in which male flowers that produce pollen and female flowers that produce seeds occur on separate individuals. Complete lack of seed production in males could result in reduced densities of conspecific seeds/seedlings and natural enemies, and thus, increased conspecific seedling recruitment, near males relative to females. On the other hand, facilitation of conspecific seedlings near reproductive females relative to males can also occur in some plant species, primarily those for which recruitment is driven primarily by abiotic rather than biotic factors (Montesinos et al. 2007). Male and female adults of dioecious species can sometimes be associated with different abiotic habitats (Bierzychudek & Eckhart 1988).

If biotic or abiotic conditions differ near male and female trees, this could influence the strength conspecific recruitment limitation. For example, if males occupy less nutrient-

rich habitats, intraspecific competition with seedling neighbors for resources could limit seedling survival more strongly near males than near females. Hood et al. (2004) conducted a study comparing conspecific seedling recruitment in the soil microbial communities of female and male adults of the dioecious tropical tree species Milicia regia (Moraceae) in Ghana, and did not find differences in seedling survival, growth, or

AMF colonization in female vs. male soil microbial communities in the shadehouse.

However, intraspecific variation in seed production remains to be examined as a factor influencing conspecific seedling recruitment in a great majority of dioecious plant species, and in field conditions in which other biotic factors, such as herbivores and neighborhood effects, can act on seedling recruitment. If seeds that are dispersed near male conspecifics are more likely to survive than seeds near females for any reason, this could give dioecious species a recruitment advantage over hermaphroditic species, helping to counteract the fact that a large proportion of dioecious individuals (i.e., males) do not produce seeds and helping maintain dioecious species in plant communities.

Mutualists, such as arbuscular mycorrhizal fungi, also play an important role in determining the recruitment success of conspecific seedlings near adult plants.

Association with mutualist soil microbes can potentially help seedlings counter conspecific distance- and density-dependent mortality due to pathogens (Liang et al.

2015, Bachelot et al. 2017). Colonization by AMF helps seedlings by increasing water and nutrient uptake (Smith & Read 2008), activating defensive pathways (Pozo & Azcón-

Aguilar 2007), and occupying colonization sites that could otherwise be occupied by pathogens (Newsham et al. 1995). Variation in seed production or soil nutrients between

males and females could also impact the density of host-specific mutualists in their environments or the level of association of mutualists with seedlings. If seed production increases host-specific AMF densities by providing additional hosts, this could potentially increase seedling colonization by AMF, and thus, increase conspecific seedling performance in female soils. However, if seed production increases densities of both AMF and pathogens in soils, AMFs might be outcompeted by pathogens, and have little effect on seedling performance. Though negative impacts of the soil microbial communities near adults on conspecific seedings appear to be more common than positive impacts, especially in AMF-associated trees (Mangan et al. 2010, Liu et al. 2012,

McCarthy-Neumann & Ibáñez 2013, Bennett et al. 2017), AMF benefits to seedlings are strong enough to balance the negative impact of host-specific pathogens in at least some tree species (Liang et al. 2015). In addition, if soil nutrient conditions vary near male versus female trees, this could lead to differences in AMF densities or interactions between seedlings and AMF near males versus females.

We conducted parallel shadehouse and field experiments to examine the impact of seed production on conspecific seedling performance near female and male adults of a dioecious tropical tree species, Virola surinamensis (Myristicaceae), in Panama. Studying a dioecious species allowed us to isolate the impact of seed production from the impact of conspecific adult presence on conspecific seedling performance. We hypothesized that female trees, which regularly input seeds beneath their canopies, reduce conspecific seedling survival and growth more strongly than male trees. The shadehouse experiment isolated the influence of male vs. female soil microbial communities on seedling growth,

as well as on colonization by arbuscular mycorrhizal fungi (AMF). In the field, we compared survival and growth of seedlings planted beneath the canopy of male and female adults, where they were naturally exposed to all potential factors affecting survival and growth, including herbivores, competition, and abiotic conditions, in addition to soil microbes. By comparing the impacts of seed production on natural-enemy mediated seedling survival and growth in both the field and the shadehouse, we hope to better understand the causes of intraspecific variation in conspecific seedling mortality near reproductive trees, and how such variation can shape tropical tree diversity and abundance.

Methods

Study site and species

We conducted our study on Virola surinamensis (Rol. ex Rottb.) Warb.

(Myristicaceae), a tropical tree species, on Barro Colorado Island (BCI), Republic of

Panama (9°09’ N, 79°51’ W). BCI is a species-rich 15.6 km2 lowland, tropical moist forest (Croat 1978). BCI is seasonally dry, receiving an average of 2623 mm of rain per year, punctuated by a distinct dry season (Windsor 1990). Virola surinamensis is a dioecious, emergent canopy species native to tropical and subtropical wet lowland forests across Central America and Amazonia, and is relatively common on BCI (Croat 1978).

On BCI, adults are most commonly found on slopes and near streams (Harms et al.

2001). Virola surinamensis adults are spatially aggregated (Condit et al. 2000, Riba-

Hernández et al. 2014), and were found to have random sex distributions and a uniform

sex ratio in Costa Rica (Riba-Hernández et al. 2014). On BCI, Flowering of V. surinamensis peaks in the dry season (~ January) and seed production peaks in the following wet season (~ July) (Zimmerman et al. 2007). In Brazil, flowers were found to be pollinated by two species of flies in the Syrphidae family (Copestylum sp. and

Erystalys sp.; Jardim & Mota 2007). Seeds are large (~2 cm), borne singly inside a woody capsule, and are surrounded by a nutritious red aril that attracts several species of large birds, such as chestnut-mandibled and keel-billed toucans (Ramphastos swainsonii and Ramphastos sulfuratus, respectively), crested guans (Penelope purpurascens), slaty- tailed trogons (Trogon massena), rufous motmots (Baryphthengus martii), and collared aracaris (Pteroglossus torquatus), as well as spider monkeys (Ateles geoffroyi), as primary seed dispersers (Howe & Vande Kerckhove 1981). On average, 62 + 5% of the seeds produced by a maternal tree are dispersed away by animals, while the remaining

37% are dropped or regurgitated under the tree (Howe & Vande Kerckhove 1981). Fruit crop size varies among females, but hundreds to thousands of seeds are normally dropped below maternal crowns (Howe et al. 1985; Eck, unpublished data). Insects and mammals kill 99.2% of seeds and/or seedlings near maternal trees within the first 12 weeks after germination (Howe et al. 1985), but recruitment success near paternal trees has not been studied. Virola surinamensis seedlings are shade-tolerant (Howe 1990), drought-sensitive

(Fisher et al. 1991), and are more likely to survive if dispersed >20 m away from the maternal tree (Howe et al. 1985).

Shadehouse experiment: male vs. female soil microbial communities

We collected 2,854 seeds from the ground beneath the canopy of 28 fruiting V. surinamensis during the peak of the species’ fruiting event (in June and July) on BCI in

2014. An average of 102 seeds were collected per tree. We assigned the fruiting tree that a seed was collected beneath as the seed’s putative mother, and kept seeds from each maternal source separate. Because V. surinamensis is dioecious, all fruiting trees were maternal parents; the paternal parentage of the seeds is not known. Trees were located by exploring a ~3.5 km2 area of BCI. After collection, seeds were surface sterilized (10% bleach for 2 min., rinse, 70% ethanol for 2 min., rinse), air dried, planted in germination trays containing autoclaved BCI soil in the shadehouse, and kept well-watered and shaded (under two layers of 80% shadecloth). Seed germination rates were low (~11%).

The maternal trees yielding > 12 seedlings healthy in appearance one month after germination were included in the experiment, for a total of 11 maternal trees and 193 experimental seedlings. We also identified four reproductive-size male V. surinamensis from within this ~3.5 km2 area. Maleness was assumed by lack of seed production in

2014, and all males were re-visited to check for seed production in 2015 and 2016. A spatial map of the location of the 11 maternal trees and 4 male trees on BCI is provided in

Figure 21. Spatial distances between pairs of adult trees range from ~30 m to ~ 2 km.

Pairs of trees that were closer together are more likely to be relatives (Riba-Hernández et al. 2014), but the level of relatedness of the adult trees and seedlings in the study to one another is not known. The adult trees were large, ranging from 50 to 100 cm DBH

(diameter at breast height); the minimum reproductive size for V. surinamensis is ~30 cm

DBH (Riba-Hernández et al. 2014).

Figure 21: A spatial map of the location of the 11 female and 4 male trees in the ‘male vs. female’ shadehouse experiment on BCI. The location of each Virola surinamensis male and female adult tree in the shadehouse experiment. Female trees, in white, are labelled V1 through V11; male trees, in yellow, are labelled M1, M2, M3, and M5. Satellite image obtained from Google Maps ® 2017.

We also collected a sample of the soil microbial community beneath the canopy of each of the adult trees in the experiment to use as inocula, for a total of 15 inocula (one per adult tree). To create inoculum for each adult tree, we collected soil at a depth of 10 cm from three randomly-selected points within 3 m of the trunk, then coarsely sieved,

combined, and homogenized the soil from the three points. All soil microbial community samples were collected in September 2014, and all inocula were created and used in the experiment within two days after field collection.

Within each of the 11 seedling cohorts, seedlings were assigned to one of two experimental treatment groups: 'male soil’ (n = 48 seedlings), or ‘female soil’ (n = 145 seedlings) (seedlings were assigned to a group at random). Each seedling in the male group was assigned to be planted in the soil microbial inoculum of a male tree in the experiment. Because maternal parentage of seedlings to female trees could be assumed from the collection method, seedlings in the female group were divided in to two smaller groups: ‘non-parent female’ or ‘parent female’. Each seedling in the parent female group

(n = 72 seedlings) was assigned to be planted in the soil microbial inoculum of its maternal parent tree. Each seedling in the non-parent female group (n = 73 seedlings) was assigned to be planted in the soil microbial inoculum of one of the female trees in the experiment that was not its parent. Thus, each seedling was assigned to be planted in the soil microbial community of only one adult tree in the experiment (either a male or a female). Seedlings in the non-parent female group were assigned at random to either female V1, V2, V3, V4, V5, or V6 (barring their own mother, if one of those trees); seedlings in the male group were assigned at random to any of the male trees (M1, M2,

M3, or M5). Sample sizes for each treatment group and each combination of maternal seed source and soil microbial inoculum source in the experiment are listed in Table 13.

Table 13: Sample sizes for each treatment group and each combination of maternal seed source and soil microbial inoculum source in the ‘male vs. female’ shadehouse experiment. The number of seedlings planted in each combination of maternal seed source and soil microbial inoculum source at the beginning of the shadehouse experiment. Rows represent maternal seed sources V1 through V11. Columns represent female soil microbial inoculum sources (V1 through V11) and male soil microbial inoculum sources (M1, M2, M3, or M5). Seedlings in the female soil microbial inoculum treatment group (n = 145 seedlings) are coded in yellow or green (green = parent female soil microbial inoculum, n = 72 seedlings; yellow = non-parent female soil microbial inoculum, n = 73 seedlings). Seedlings in the male soil microbial inoculum treatment group are coded in gray (n = 48 seedlings). Dashes in cells indicate lack of replicates for a seed source x soil source combination.

two layers of 80% shade cloth (to mimic a shady understory) and was shielded from rainfall with a roof of clear plastic lining. Initial stem height, leaf number, and leaf area

(length and width) of each seedling was measured immediately after transplant. Seedlings remained in their experimental treatments for eight months, during which time seedling survival and growth (stem height, leaf number, and leaf length and width) were measured periodically (every 2-4 weeks), and each seedling was watered three times per week (see

Fig. 3 for a photograph of the set-up in the shadehouse). After 8 months, the 183 surviving seedlings were harvested (i.e., gently dug from their pots and their roots washed to remove soil) to obtain measurements of final oven-dried biomass, stem height, and leaf area, and to collect fine root samples for the AM fungi analysis. Fine root samples were stored in 70% ethanol and refrigerated until being shipped to Yale

University (USA) for AM fungi quantification.

Figure 22: A photograph of the ‘male vs. female’ shadehouse experiment. The photograph shows V. surinamensis seedlings growing individually in pots on a shadehouse bench. Pots contained the soil microbial community of either an adult male or an adult female tree in the population.

Quantification of colonization by AM fungi in shadehouse seedlings

To quantify colonization by AM fungi in the seedlings that survived the shadehouse experiment, we used a magnified root intersect method (McGonigle et al.

1990). To prepare roots for quantification, we used a modified root clearing and staining protocol (Giovannetti and Mosse 1980; McGonigle et al. 1990; Vierheilig et al. 2005;

INVAM 2015). For each seedling, we cleared a random 0.2 mg subsample of the seedling’s fine root mass in 10% bleach, 10% KOH, and 1% HCL. We then stained the subsamples using direct blue stain and mounted them on glass slides using lactic acid.

We quantified the colonization of AM fungi in the subsamples at 200x magnification under a compound light microscope by recording the presence or absence of visible AM fungi structures (hyphae, vesicles, arbuscules, or arbuscular coils) for a minimum of 35 root intersects per seedling (see Figure 23 for a micrograph of the structures quantified).

We obtained high-quality AM fungi colonization data for 149 of the 183 surviving experimental seedlings. We calculated AM fungi colonization for each of these seedings as the proportion of root intersects quantified in the subsample that contained any visible

AM fungi structure. We also calculated the proportion of root intersects quantified that contained visible arbuscules only, as a more conservative estimate of colonization by AM fungi.

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Figure 23: A micrograph of AM fungi structures in the root of a seedling in the ‘male vs. female’ shadehouse experiment. A photograph taken at 200x magnification shows examples of each of the four AM fungi structures quantified in the AM fungi study (hyphae, arbuscules, vesicles, and coils) stained in blue inside the root of an experimental seedling.

Quantification of seed production of the female trees in the shadehouse experiment

We also quantified the number of seeds produced by 9 of the 11 females in the shadehouse experiment during the 2014 seed production event. Seed production could not be accurately quantified for two females located on disturbed edges of the station clearing

(V7 and V8). V. surinamensis seeds are enclosed by a heavy, woody, inedible capsule whose two halves dehisce to reveal a single seed. Capsules are not dispersed and fall to the ground beneath the canopy, where they take several months to deteriorate. Capsules can be counted after the end of a fruiting period to quantify total seed production during 101

the period. Using the method of Queenborough et al. 2007b, we first calculated the total canopy area of each female by measuring the distance to the edge of the canopy in the four cardinal and four intercardinal directions from the trunk, drawing an octagon from the canopy edge points, then summing the area of the eight triangles within the octagon to estimate the total canopy area. After the 2014 seed production period, we counted all entire seed capsules and capsule halves found within a randomly selected sample of

~10% of each tree’s canopy area, then calculated the number of capsules (counting every two capsule halves found as an additional capsule) in the sample area of each tree. Using the number of capsules in the sample area of each tree, we estimated the number of capsules in the total canopy area of each tree, which is equal to the total number of seeds produced by each tree in the seed production event.

Field experiment: male vs. female environments

We collected 3,581 seeds from the ground beneath the canopy of 23 fruiting female trees of V. surinamensis on BCI in June and July 2015. Trees were located by exploring the same ~3.5 km2 area of BCI as in the shadehouse study. The average number of seeds collected per tree was 156. After collection, all seeds were surface sterilized (10% bleach for 2 min., rinse, 70% ethanol for 2 min., rinse), air dried, planted in germination trays containing autoclaved BCI soil in the shadehouse, and kept well- watered under two layers of 80% shadecloth. Seed germination rates were low (~7%).

The 6 female trees providing sufficient numbers of seedlings healthy in appearance one month after germination were selected for use in the experiment, for a total of 187

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experimental seedlings. Because of annual variation in seed production and/or germination success, only 2 of the female V. surinamensis studied in the shadehouse experiment could be included in the field experiment (V5 and V11) the other 4 female trees in the field experiment were not studied in the shadehouse. The same 4 reproductive-size male V. surinamensis studied in the shadehouse experiment were also selected for study in the field experiment (M1, M2, M3, and M5). A spatial map of the location of the 6 female and 4 male trees on BCI is provided in Figure 24. Spatial distances between pairs of adult trees range from ~30 m to ~2 km.

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Figure 24: A spatial map of the location of the 6 female and 4 male trees in the male vs. female field experiment on BCI. The location of each male and female Virola surinamensis adult in the field experiment. Female trees are shown in white (V5, V11, V12, V13, V14, V15); male trees are shown in yellow (M1, M2, M3, and M5). Satellite image obtained from Google Maps ® 2017.

Seedlings were assigned to one of two experimental field treatment groups: ‘male conspecific environment’ or ‘female conspecific environment’. Seedlings in the male group (n = 42 seedlings) were assigned for transplant beneath the canopy of one of the four male trees in the experiment. Seedlings in the female group (n = 145 seedlings) were assigned for transplant beneath the canopy of one of the 6 females in the experiment: either their own mother tree (n = 65 seedlings), or a female tree that was not their mother

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(n = 80 seedlings). Seedlings in the male conspecific group were assigned at random to either male M1, M2, M3, or M5; seedlings in non-mother sub-group of the female group were assigned at random to one of the five female trees that was not their mother. All seedlings were randomly assigned to one of several 1 m2 seedling plots beneath the canopy of their assigned tree. Plot placement beneath a tree’s canopy was randomized with respect to direction and distance from the base of the tree (1-4 m). Trees had between three and six plots beneath their canopies, with each plot containing between three and five experimental seedlings, depending on seedling availability. Female trees had, on average, more plots per tree and more seedlings per plot than male trees (average plots per tree = 5.2 for female trees and 3.0 for male trees; average seedlings per plot =

4.7 for female trees and 3.6 for male trees). Density was therefore included as a covariate in all statistical analyses (see below). Variation in the average number of plots and seedlings per plot between male and female trees arose due to the need for female plots to contain additional seedlings for the ‘offspring vs. non-offspring’ experimental comparison described in Chapter 2, and was not intended to be an independent variable of focus in this experiment. Seedlings in each plot were randomly assigned to one of sixteen 25 cm2 positions within the plot. Sample sizes for the field experimental treatment groups can be found in Table 14.

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Table 14: Sample sizes for each treatment group and each combination of maternal seed source and focal adult environment in the ‘male vs. female’ field experiment. The number of seedlings planted in each combination of maternal seed source and focal adult environment at the beginning of the field experiment. Rows represent maternal seed sources (V5 and V11-V15). Columns represent female adult environments (V5 and V11- V15) and male adult environments (M1, M2, M3, or M5). Seedlings in the female environment treatment group (n = 145 seedlings) are coded in yellow or green (green = mother environment, n = 65 seedlings; yellow = other female environment, n = 80 seedlings). Seedlings in environments are in gray (n = 43 seedlings). Empty cells indicate a lack of replicates for the seed source x focal adult combination.

All experimental seedlings were transplanted into their experimental plots in the field in August 2015, at ~1 month of age. At the time of transplant, seedlings were tagged with a unique identification number and initial stem height, leaf number, and leaf area

(length and width) were measured. Seedlings were censused every 1-2 months, during which their status (alive/dead) was recorded and stem height and leaf number measured

(for the surviving seedlings) (see Figure 25 for a photograph of the field set-up). We also recorded the status of physical damage to the seedlings, such as stem breakage (likely by agoutis or other mammalian herbivores), as ‘clipping’ (clipped/unclipped). Clipping was common – 61 out of the 187 experimental seedlings (32.6%) were observed to be clipped at some point during the experiment. Due to high rates of clipping in the field 106

experiment, we included clipped status as a covariate in the models of seedling growth and survival, and also modelled the predictors of clipping (Appendix D). In addition, some seedlings were uprooted between censuses (perhaps by peccaries), and were found lying nearby (18 seedlings, 9.6%) – uprooted seedlings were removed from all statistical analyses. Some seedlings decayed, were destroyed, or otherwise disappeared between censuses, meaning no remnants of the dead seedling could be found (but their tags usually were found). The clipping and/or uprooting status of these seedlings could not be determined, meaning that actual clipping or uprooting rates could be higher than we observed. After ~8 months (in March 2016), the surviving seedlings (31 out of 187 seedlings, 16.6%) were harvested and brought to the lab, where their total dry biomass, stem height, and leaf area were measured. Low survival rates could be due partially to drought: the final months of the field experiment overlapped with a severe dry season due to the strong 2015/16 El Niño event, and signs of wilting were observed in some seedlings.

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Figure 25: Censusing seedling survival and biomass in the field plots. Research assistant Lourdes Hernández measures tagged experimental seedlings in a field plot on BCI. Orange flags mark the corners of the 1 m2 plot.

Models of seedling biomass and colonization by AM fungi in the shadehouse

We analyzed the effect of the experimental shadehouse treatment (i.e., inoculation with the soil microbial community of a male conspecific or a female conspecific) on seedling total dry biomass at harvest using a linear mixed-effects model. We focused on biomass (i.e., growth) because survival was high in the shadehouse (94.8%, 183 out of

193 seedlings). Dead seedlings were not included in any shadehouse analyses. To account for potential sources of variation in seedling biomass, such as variation among seedling cohorts (e.g., maternal effects), or in the biotic or abiotic conditions found among soil microbial inocula or among benches in the shadehouse, maternal seed source, soil

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microbial inoculum source, and shadehouse bench were included as random effects in the model. To account for the potential impact of variation in the size of the seedlings at the beginning of the experiment, we also included an estimate of initial seedling dry biomass as a covariate in the model. Initial dry biomass was estimated for each experimental seedling based on height at the time of transplant using an allometric linear regression model based on measurements of height and biomass of a randomly harvested sample of

-16 2 the potential experimental seedlings (F1,42 = 338.1, p < 2.2e , R = 0.89). Because we found a difference in seedling biomass in mother vs. other female soil microbial communities in Chapter 1, we then constructed a model with identical terms that excluded those seedlings planted in their own mother’s soil microbial community, to compare seedling biomass in male and female soil microbial communities when neither adult is a known parent of the seedling.

We also analyzed the effect of the experimental shadehouse treatment on the proportion colonization by AM fungi in seedlings at the end of the experiment using a generalized linear mixed-effects model with binomial errors. As above, estimated initial seedling dry biomass was included as a covariate, and maternal seed source, soil microbial inoculum source, and shadehouse bench were included as random effects in the model. To account for potential variation between the two researchers that quantified AM fungi colonization in the seedlings, we included observer as a fixed effect in the model.

Because we also found a difference in seedling colonization by AM fungi in mother vs. other female soil microbial communities in Chapter 1, we then constructed a model with identical terms that excluded those seedlings planted in their own mother’s soil microbial

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community, to compare seedling colonization by AM fungi in male and female soil microbial communities when neither adult is a known parent of the seedling.

We also analyzed the effect of seed production among nine of the 11 females in the shadehouse experiment on seedling total dry biomass and proportion colonization by

AM fungi in those female’s soil microbial communities. Seedling biomass was analyzed as a function of the log-transformed number of seeds produced by the female whose soil it was planted in using a linear mixed-effects model. Proportion colonization by AM fungi was analyzed as a function of the log-transformed number of seeds produced by the female whose soil it was planted in using a generalized linear mixed-effects model with binomial errors. In both models, we also included initial seedling biomass as a fixed effect, as well as maternal seed source, soil microbial inoculum source, and shadehouse bench as random effects. Observer was included as a fixed effect in the AM fungi model.

Models of seedling survival and biomass in the field

We analyzed the effect of the experimental field treatment (i.e., planting beneath the canopy of a male conspecific or a female conspecific) on seedling survival to harvest using a generalized linear mixed-effects model with binomial errors. In this model, the number of conspecific seedlings in the seedling’s plot was included as a fixed effect. We also tried including an interaction term, allowing for an interaction between treatment and the number of conspecific seedlings, but this term was not significant, and was not included in the model. To account for other potential sources of variation in seedling survival, we included maternal seed source, focal adult tree (i.e., transplant site), and plot

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as random effects in the model. To account for the potential impact of variation in the size of the seedlings at the beginning of the experiment, we also included an estimate of initial seedling dry biomass as a covariate in the model. Initial dry biomass was estimated for each experimental seedling based on height and estimated leaf area (based on the measurements of leaf length and width) at the time of transplant using an allometric linear regression model based on measurements of the height, leaf area (measured with a leaf area machine), and biomass of a randomly harvested sample of the potential

2 experimental seedlings (F2,47 = 496.5, p < 0.001, R = 0.95). We also included clipping during the experiment (clipped/unclipped) as a binary fixed effect in the model. To ensure that variation in the number of conspecific seedlings in male and female plots was not causing problems with model interpretation (e.g., due to violations of the assumption of independence), we also constructed a similar model comparing seedling survival in male and female environments in plots with four seedlings only, for comparison. Also, because we found a difference in seedling growth in mother vs. other female soil microbial communities in Chapter 1, we also constructed a model with identical terms that excluded those seedlings planted near their own mother.

We also analyzed the effect of the experimental field treatment (i.e., planting beneath the canopy of a male conspecific or a female conspecific) on seedling total dry biomass at harvest using a linear mixed-effects model. Dead seedlings were not included in the biomass analysis. As above, the number of conspecific seedlings in the seedling’s plot and initial seedling biomass were included as fixed effects in the model, and maternal seed source, focal adult tree, and plot were included as random effects. The

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interaction between treatment and the number of conspecific seedlings was included initially but was dropped due to non-significance. As above, we also constructed a similar model comparing seedling biomass in male and female environments in plots with four seedlings only. Again, because we found a difference in seedling biomass in mother vs. other female soil microbial communities in Chapter 1, we also constructed a model with identical terms that excluded those seedlings planted near their own mother.

All mixed-effects analyses in our study were performed using the lme4 package

2016). ANOVA tables for the mixed-effects models were obtained with the afex package

(Singmann et al. 2017).

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Results

Biomass of seedlings in the shadehouse

We found that total biomass was similar for seedlings growing in male and female soil microbial communities (Table 15 & Fig. 26). Six out of the 10 maternal seed sources tested had lower mean biomass in female soil microbial communities than in male soil microbial communities (Figure 27), but this difference was not statistically significant at the treatment level. Removing the seedlings planted in their own mother’s soil microbial community from the analysis did not change the lack of a significant effect of treatment

(Appendix E, Table 27). We also did not find an effect of seed production on seedling biomass in female soil microbial communities (Table 16 & Figure 28). Most seedlings grew substantially during the experiment: average total biomass at the end of the experiment was 2.58 g, while average total biomass at the beginning of the experiment was estimated at 0.39 g. Seedlings that were larger at the beginning of the experiment

(i.e., those with higher initial biomass) had significantly higher final biomass.

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Table 15: Mixed-model ANCOVA of biomass of seedlings in male and female soil microbial communities in the shadehouse. ANCOVA table of the predictors of total dry biomass of V. surinamensis seedlings in male vs. female soil microbial communities at the end of the 8-month soil microbial inoculation shadehouse experiment (n = 183 seedlings). Intercept = seedlings grown in the female soil microbial communities; Male soil microbial communities = seedlings grown in male soil microbial communities. Initial biomass = a seedling’s estimated biomass at the beginning of the experiment. Fixed effects summary includes coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), t-score (t = β/SE(β)), F-value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (n = 11 maternal seed sources, 15 soil microbial inoculum sources, and 4 shadehouse benches).

Fixed Effects β SE(β) df t F p

Intercept 0.91 0.39 5.52 2.33 NA 0.06

Male soil microbial 0.21 0.22 11.50 0.96 0.92 0.36 community

Initial Biomass 4.08 0.42 172.13 9.67 93.60 < 2e-16

Random Effects Var. SD Maternal Seed Source 0.05 0.22 Soil Microbial Inoculum Source 0.08 0.29 Bench 0.43 0.66 Residual 0.57 0.75

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Figure 26: Biomass of seedlings in male vs. female soil microbial communities in the shadehouse. The total biomass (g) of V. surinamensis seedlings grown in male vs. female soil microbial communities at the end of the 8-month shadehouse experiment (n = 145 seedlings in female soil microbial communities; n = 42 seedlings in male soil microbial communities). A linear mixed-effects model revealed that seedlings had similar biomass in male and female soil microbial communities (p = 0.36). Whiskers are + 1.5*IQR.

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Figure 27: Biomass of seedlings across 11 maternal seed sources in male vs. female soil microbial communities in the shadehouse. The total biomass of seedlings from 11 maternal seed sources in male vs. female soil microbial communities (n = 183 seedlings). Median biomass was lower in female soil microbial communities relative to male soil microbial communities in 6 out of the 10 maternal seed sources tested (NA represents missing data). Whiskers are + 1.5*IQR.

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Table 16: Mixed-model ANCOVA of the effect of seed production on seedling biomass in female soil microbial communities in the shadehouse. ANCOVA table of seed production and initial biomass as predictors of total biomass of V. surinamensis seedlings in female soil microbial communities at the end of the 8-month shadehouse experiment (n = 130 seedlings). Intercept = seedlings grown in a female soil microbial community with no seed production; seed production = the impact of adding one unit of log-transformed seed production; initial biomass = the impact of a seedling’s estimated biomass at the beginning of the experiment. Fixed effects summary includes coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), t-score (t = β/SE(β)), F- value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (i.e., 11 maternal seed sources, 9 female soil microbial inoculum sources, and 4 shadehouse benches).

Fixed Effects β SE(β) df t F p

Intercept 2.34 1.05 8.91 2.24 NA 0.05

Seed Production -0.16 0.12 7.13 -1.33 1.75 0.23

Initial Biomass 3.81 0.46 120.10 8.35 69.74 1.36e-13

Random Effects Var. SD Maternal Seed Source 0.10 0.31 Soil Microbial Inoculum Source 0.02 0.16 Bench 0.46 0.68 Residual 0.48 0.69

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Figure 28: Seedling biomass in the soil microbial communities of females that varied in seed production. The total dry biomass of seedlings in the soil microbial communities of female conspecifics that varied in seed production at the end of the 8-month shadehouse experiment (n = 130 seedlings). A linear mixed-effects model revealed that there was not a significant relationship between log-transformed seed production and biomass (p = 0.22).

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Colonization by AM fungi of seedlings in the shadehouse

We found that colonization by AM fungi was similar for seedlings growing in male and female soil microbial communities (Table 17 and Figure 29). Seven out of the

10 maternal seed sources tested had lower mean proportion colonization by AM fungi in female soil microbial communities than in male soil microbial communities (Figure 30), but this difference was not statistically significant at the treatment level. Seedlings that were larger at the beginning of the experiment (i.e., those with higher initial biomass) had significantly higher colonization proportions. The two observers who quantified colonization proportion also varied significantly in their measurements. Removing the seedlings planted in their own mother’s soil microbial community from the analysis did not change the lack of a significant effect of treatment, but initial biomass no longer predicted proportion colonization for these non-offspring seedlings (Appendix E, Table

28). We also did not find an effect of seed production on seedling colonization by AM fungi in female soil microbial communities (Table 18 & Figure 31).

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Table 17: Mixed-model ANCOVA of colonization by AM fungi of seedlings in male and female soil microbial communities in the shadehouse. ANCOVA table of the predictors of proportion colonization by AM fungi of V. surinamensis seedlings in male vs. female soil microbial communities at the end of the 8-month soil microbial inoculation shadehouse experiment (n = 149 seedlings). Intercept = seedlings grown in female soil microbial communities and quantified by observer 1; male soil microbial communities = seedlings grown in male soil microbial communities; initial biomass = a seedling’s estimated biomass at the beginning of the experiment; observer = seedlings quantified by observer 2. Fixed effects summary includes logit-linked coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), z-score (z = β/SE(β)), F- value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (n = 11 maternal seed sources, 15 soil microbial inoculum sources, and 4 shadehouse benches).

Fixed Effects β SE(β) df z F p

Intercept -2.34 0.39 NA -6.02 NA 1.74e-09

Male soil microbial 0.62 0.56 12.16 1.11 1.24 0.27 community

Initial Biomass 1.41 0.19 109.63 7.34 101.91 2.15e-13

Observer 0.82 0.05 141.79 15.72 248.86 < 2e16

Random Effects Var. SD Maternal Seed Source 0.10 0.32 Soil Microbial Inoculum Source 0.89 0.95 Bench 0.20 0.45

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Figure 29: Colonization by AM fungi of seedlings in male vs. female soil microbial communities. The proportion of colonization by AM fungi in the roots of seedlings in male vs. female soil microbial communities at the end of the 8-month shadehouse experiment (n = 149 seedlings). A generalized linear mixed-effects model revealed that seedlings had similar colonization by AM fungi in male and female soil microbial communities (p = 0.27). Whiskers are + 1.5*IQR.

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Figure 30: Colonization by AM fungi of seedlings across 11 maternal seed sources in male vs. female soil microbial communities. The proportion colonization by AM fungi of seedlings from 11 maternal seed sources in male vs. female soil microbial communities (n = 183 seedlings). Mean AM fungi colonization proportion was lower in female soil microbial communities relative to male soil microbial communities in 7 out of the 10 maternal seed sources tested (NA represents missing data). Whiskers are + 1.5*IQR.

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Table 18: Mixed-model ANCOVA of the effect of seed production on seedling colonization by AM fungi in female soil microbial communities in the shadehouse. ANCOVA table of seed production and initial biomass as predictors of proportion colonization by AM fungi of V. surinamensis seedlings in female soil microbial communities at the end of the 8-month shadehouse experiment (n = 105 seedlings). Intercept = seedlings grown in a female soil microbial community with no seed production and quantified by observer 1; seed production = the impact of adding one unit of log-transformed seed production; initial biomass = the impact of a seedling’s estimated biomass at the beginning of the experiment; observer = seedlings quantified by observer 2. Fixed effects summary includes logit-linked coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), z-score (z = β/SE(β)), F-value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (n = 11 maternal seed sources, 9 female soil microbial inoculum sources, and 4 shadehouse benches).

Fixed Effects β SE(β) df z F p

Intercept -8.08 4.31 NA -1.87 NA 0.06

Seed Production 0.68 0.53 5.95 1.30 2.27 0.20

Initial Biomass 1.41 0.21 88.36 6.59 82.36 4.27e-11

Observer 0.86 0.06 99.32 13.58 185.10 < 2e-16

Random Effects Var. SD Maternal Seed Source 0.27 0.52 Soil Microbial Inoculum Source 1.41 1.19 Bench 0.15 0.39

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Figure 31: Seedling colonization by AM fungi in the soil microbial communities of females that varied in seed production. The colonization by AM fungi of seedlings in the soil microbial communities of female conspecifics that varied in seed production at the end of the 8-month shadehouse experiment (n = 105 seedlings). A generalized linear mixed-effects model revealed that there was not a significant relationship between log- transformed seed production and colonization by AM fungi (p = 0.20).

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Survival of seedlings in the field experiment

We found that the probability of survival was similar for seedlings near male conspecifics and near female conspecifics in the field (Table 19 & Figure 32). Four out of the six maternal seed sources tested had lower mean probabilities of survival in female environments than in male environments (Figure 33), but this difference was not statistically significant at the treatment level. Seedling survival tended to decrease as the number of conspecific seedlings in a plot increased, but this effect was not quite statistically significant (Figure 34). Seedlings that were larger at the beginning of the experiment (i.e., those with higher initial biomass) had a higher probability of survival.

Clipping did not influence seedling survival. We did not find that the type of environment a seedling was in (male or female), the number of conspecific seedling neighbors, or the initial biomass of a seedling influenced the probability the seedling would be clipped

(Appendix D, Table 26). Removing clipped seedlings from our analyses did not result in changes to our findings. Removing the seedlings planted in their own mother’s soil microbial community from the analysis did not change the lack of a significant effect of treatment on survival, but strengthened the negative impact of conspecific seedlings on survival (Appendix E, Table 29). Analyzing only the seedlings planted in plots with four conspecific seedlings also did not change the results of this model (Appendix C, Table

24).

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Table 19: Mixed-model ANCOVA of survival of seedlings in male and female environments in the field. ANCOVA table of the predictors of V. surinamensis seedling survival in male vs. female environments at the end of the 7-month field experiment (n = 169 seedlings). Intercept = the probability of survival of unclipped seedlings in female environments; male environment = the impact of being in a male environment on the seedling survival; number of conspecific seedlings in a plot = the impact of adding one conspecific seedling to the plot on seedling survival; clipping = the impact of being clipped on seedling survival; initial biomass = the impact of initial biomass on seedling survival. Fixed effects summary includes logit-linked coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), z-score (t = β/SE(β)), F-value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (i.e., 6 maternal seed sources, 10 focal adults, and 43 seedling plots).

Fixed Effects β SE(β) df z F p

Intercept 0.62 3.24 NA 0.19 NA 0.85

Male Environment -0.49 1.73 15.28 -0.27 0.42 0.79

Number of Conspecific -1.28 0.67 39.99 -1.90 1.77 0.06 Seedlings in a Plot

Clipping -0.78 0.70 158.74 -1.128 0.78 0.26

Initial Biomass 8.75 2.49 63.61 3.52 21.18 4.35e-04

Random Effects Var. SD Maternal Seed Source 0.40 0.63 Focal Adult 4.42 2.10 Plot 0.32 0.57

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Figure 32: Seedling survival near male vs. female conspecifics in the field. Fitted survival probabilities for seedlings near a female conspecific adult vs. seedlings near a male conspecific adult at the end of the 8-month field transplant experiment (n = 169 seedlings). A generalized linear mixed-effects model revealed that there was not a significant effect of conspecific environment on seedling survival (p = 0.79). Whiskers are + 1.5*IQR.

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Figure 33: Survival of seedlings across 6 maternal seed sources in male vs. female environments in the field. The fitted probability of survival of seedlings from 6 maternal seed sources in male vs. female environments in the field (n = 169 seedlings). Survival probabilities were lower in female environments relative to male environments in 4 out of the 6 maternal seed sources tested. Whiskers are + 1.5*IQR.

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Figure 34: Seedling survival near male vs. female conspecifics in the field depending on conspecific seedling density. Fitted probabilities of survival for seedlings near a female conspecific adult (in blue) vs. seedlings near a male conspecific adult (in pink) at the end of the 8-month field transplant experiment, shown across the number of conspecific seedlings in a seedling’s plot (n = 169 seedlings). There was a marginally significant effect of the number of conspecific seedling neighbors on seedling survival (p = 0.06).

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Biomass of seedlings in the field

We found that biomass was similar for seedlings that survived near male conspecifics and seedlings that survived near female conspecifics in the field (Table 20 &

Figure 35). Three out of the four maternal seed sources tested had higher mean biomass in female environments than in male environments (Figure 36), but this difference was not statistically significant at the treatment level (missing values due to dead seedlings prevented comparison for the other 2 seed sources in the field experiment). The number of conspecific seedlings in a plot did not influence seedling biomass (Figure 37).

Seedlings that were larger at the beginning of the experiment (i.e., those with higher initial biomass) had higher biomass. Clipped seedlings had significantly lower biomass than unclipped seedlings. Removing the seedlings planted in their own mother’s soil microbial community from the analysis did not change the lack of a significant effect of treatment on biomass (Appendix E, Table 30). Analyzing only the seedlings planted in plots with four conspecific seedlings also did not change the results of this model

(Appendix C, Table 25).

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Table 20: Mixed-model ANCOVA of the biomass of seedlings in male and female environments in the field. ANCOVA table of the predictors of V. surinamensis seedling biomass in male vs. female environments at the end of the 7-month field experiment (n = 31 seedlings). Intercept = the biomass of unclipped seedlings in female environments; male environment = the impact of being in a male environment on seedling biomass; number of conspecific seedlings in a plot = the impact of adding one conspecific seedling to the plot on seedling biomass; clipping = the impact of being clipped on seedling biomass; initial biomass = the impact of initial biomass on final seedling biomass. Fixed effects summary includes coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), t-score (t = β/SE(β)), F-value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (i.e., 5 maternal seed sources, 8 focal adults, and 22 seedling plots).

Fixed Effects β SE(β) df t F p

Intercept 0.77 0.43 17.62 1.79 NA 0.09

Male Environment -0.04 0.15 15.10 -0.23 0.05 0.82

Number of Conspecific -0.07 0.09 17.47 -0.73 0.53 0.48 Seedlings in a Plot

Clipping -0.26 0.06 11.90 -4.29 18.36 1.08e-02

Initial Biomass 0.38 0.16 11.20 2.42 5.85 0.03

Random Effects Var. SD Maternal Seed Source 0.000 0.000 Focal Adult 0.003 0.054 Plot 0.037 0.192 Residual 0.008 0.091

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Figure 35: Seedling biomass near male vs. female conspecifics in the field. Biomass of seedlings near female adults vs. male adults at the end of the 7-month field experiment (n = 31 seedlings). A linear mixed-effects model revealed that there was not a significant effect of conspecific environment on seedling biomass (p = 0.82). Whiskers are + 1.5*IQR.

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Figure 36: Biomass of seedlings across 6 maternal seed sources in male vs. female environments in the field. The fitted biomass of seedlings from 6 maternal seed sources in male vs. female environments in the field (n = 31 seedlings). Median biomass was higher in female environments relative to male environments in 3 out of the 4 maternal seed sources where data for comparisons were available. Whiskers are + 1.5*IQR.

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Figure 37: Seedling biomass near male vs. female adults in the field across conspecific seedling densities. Biomass of seedlings near a female conspecific adult (in blue) vs. seedlings near a male conspecific adult (in pink) at the end of the 8-month field transplant experiment, shown across the number of conspecific seedlings in a seedling’s plot (n = 31 seedlings). A linear mixed-effects model revealed that there was not a significant effect of the number of conspecific seedling neighbors on seedling survival (p = 0.48).

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Discussion

In parallel shadehouse and field experiments, we did not find evidence in support of the hypothesis that seed production by females of the dioecious tropical tree species

Virola surinamensis decreases conspecific seedling performance near female adults relative to male adults. We found that seedling growth and colonization by AMF were similar in male and female soil microbial communities in the shadehouse. This indicates that seed input into soils by females during a fruiting event does not affect seedling performance in those soils. In addition, we did not find a relationship between the amount of seed input in female soils and seedling growth or colonization by AMF in those soils in the shadehouse. In the field, where seedlings were exposed to all potential natural enemies and variable environmental conditions, seedling survival and growth over the cross of the experiment were also similar for seedlings growing beneath the canopy of a seed-producing female and for seedlings growing beneath the canopy of a non-seed producing male. Overall, our experiments show that seed input in to the environments near female trees does not decrease early conspecific seedling performance near females relative to males in this species.

The concept that seed input into the environments near adult plants during seed production promotes the accumulation of host-specific natural enemies is intuitive, as conspecific seeds and seedlings represent additional resources for host-specific natural enemies to grow and reproduce. Though we did not find indirect evidence for the hypothesis that seed production causes the accumulation of host-specific natural enemies by examining seedling performance, our experiments do not rule out the possibility that

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seed input could cause short- or long-term changes to the density or composition of natural enemy communities near fruiting plants. They do suggest, however, that any such changes, if they do occur, do not have a large influence on conspecific seedling recruitment during the first year, at least in our study species. Significant effects of seed production on conspecific seedling recruitment via natural enemies could still emerge over longer time periods, during later developmental stages, or could be critically determined during or immediately after seed germination. We suggest that future studies test explicitly for short- and long-term changes in natural enemy composition or density near adults due to seed production.

Our experiments suggest that any changes to the soil microbial communities or general environment near female conspecific adults due to seed production do not cause conspecific seedling recruitment to differ near females relative to males. Our findings were consistent with the findings of Hood et al. (2004), who also found no differences in seedling survival, growth, or AMF colonization in female versus male soil microbial communities in the shadehouse in a dioecious tropical tree species in Ghana. The fact that seedlings in our study that were larger at the beginning of the experiment had higher colonization proportions indicates that larger seedlings either already had more AM fungi at the start of the experiment, or more easily acquired AM fungi during the experiment

(seedlings were germinated in a sterile soil medium, limiting the possibility of the former). Our study went beyond Hood et al. (2004) by also confirming lack of a difference in conspecific seedling recruitment near males and females in the field, where other natural enemies (such as herbivores and foliar pathogens) and environmental

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factors were at play. Because conspecific seedling recruitment was similar near female and male adults, this suggests that the other food resources that adult trees provides to natural enemies (e.g. leaves, roots, or wood) are more important in ultimately determining conspecific seedling recruitment than seeds (a relatively ephemeral resource in our study species). A study of several subtropical tree species in China found a positive relationship between conspecific adult density and soil pathogen density that led to increased conspecific seedling mortality (Liang et al. 2016), thus the presence and density of conspecific adults may have a larger effect on natural enemy densities than seed production. The generality of our findings will be revealed by studies examining the impact of seed production on conspecific seedling recruitment in additional plant species and communities.

Our findings add to the discussion of the advantages and disadvantages of dioecy and how dioecious species are maintained in tropical tree communities (Bruijning et al.

2017) by suggesting that males do not provide better sites for conspecific seedling recruitment than females. Though sex ratios in dioecious tree species tend to be male- biased (Queenborough et al. 2007a), the sex ratio is uniform in our study species. Adults of V. surinamensis tend to be spatially aggregated, with equal ratios of males and females in clumps (Riba-Hernandez et al. 2014). The relative tolerance of seedlings near females to any changes to female environments due to seed production could help seedlings withstand the impacts of aggregated seed production and could be a form of compensatory seedling recruitment (Queenborough et al. 2009). In addition, any environmental differences in the habitats that males and females occupy do not appear to

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have a strong effect on conspecific seedling performance. The environments near female and male adult V. surinamensis produced similar conspecific seedling performance dynamics, meaning that all adults were equal in their contribution to Janzen-Connell dynamics despite stark differences in seed production between males and females.

Our study adds to the body of literature examining the relative importance of conspecific adult neighbors vs. conspecific seedling neighbors on seedling survival

(Queenborough et al. 2007b, Comita et al. 2010, Lebrija-Trejos et al. 2014). Though conspecific seedling neighbors are known to contribute to negative density-dependent seedling survival in tropical trees (Harms et al. 2000, Wright et al. 2005, Queenborough et al. 2007, Comita et al. 2010, Metz et al. 2010, Lebrija-Trejos et al. 2014), we did not find that variation in the number of conspecific seedling neighbors influenced seedling survival or biomass in our study. This could be due to the relatively small range of neighbors utilized in our study (either 2, 3, or 4 neighbors), which might not accurately or meaningfully represent typical conspecific seedling densities in nature for our focal species. Conspecific seedlings could occur near males due to seed dispersal (especially if dispersers use male trees for food or habitat), or if males occur near fruiting females.

Our study offers a comparison of seedling survival and biomass of V. surinamensis in the field, when exposed to many potential natural enemies and variation in environmental conditions, and in the shadehouse, when exposed only to soil microbial biota in controlled environmental conditions. Very little mortality and a moderate amount of growth occurred for plants in the shadehouse, whereas mortality levels in the field were high and little growth occurred. Large decreases in seedling survival and biomass in

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the field relative to the shadehouse were likely due to herbivores or environmental stressors, such as light and drought. Clipping, likely by agoutis or other mammalian herbivores, was relatively common in the field and caused significant reductions in seedling biomass, introducing noise into our field data that could potentially be obscuring treatment effects. Light and water limitation also likely played a large role on survival and biomass in the field. Even though light levels for plants in the shadehouse were reduced by shade cloth, light levels were still likely higher on our shadehouse benches than under adult trees in the forest. In addition, plants were well watered throughout the shadehouse study, unlike in the field experiment, where seedlings experienced a severe drought and wilting was observed in some seedlings.

Understanding the biotic and abiotic factors that contribute to the accumulation of natural enemies and conspecific recruitment limitation near adult plants, as well as the sources and consequences of variation in these processes, will help us better understand how natural enemies structure plant diversity and abundance. Variation in conspecific seedling recruitment near reproductive plants within species could help to enhance plant species diversity by enabling species to differ in the distributions of their responses to the environment (Clark 2010). We suggest that future studies also examine the impact of factors other than seed production that could be important in contributing to the accumulation of natural enemies and subsequent reductions in conspecific seedling recruitment near parent plants, such as adult size, neighborhood effects, or abiotic effects.

Intraspecific variation in conspecific seedling recruitment near adult plants remains an

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important and largely unexplored consideration that could be important in structuring diversity and abundance in plant communities.

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Appendix A: AM Fungi (Chapter 1)

Standardization of proportion colonization by AM fungi

To standardize proportion colonization by AM fungi to correct for differences in

AM fungi quantification between the two observers in the experiment, we first constructed a generalized linear mixed-effects model examining the effect of observer on proportion colonization by AM fungi (with maternal seed source, soil source, and shadehouse bench as random effects) (Table 21).

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Table 21: Generalized linear mixed-effects model summary of the effect of observer on proportion colonization by AM fungi. Fixed effects summary includes logit-linked coefficient estimates (β), standard errors (SE(β)), z-score (z = β/SE(β)), and significance level (p) of each fixed effect in the model. Observer CSD = the estimate for proportion colonization of seedlings quantified by CSD. Observer JLE = the estimate for proportion colonization of seedlings quantified by JLE. Random effects summary includes variance and standard deviation of each random effect in the model (11 maternal seed sources, 11 soil microbial inoculum sources, and 4 shadehouse benches).

Fixed Effects β SE(β) z p Intercept (Observer CSD) -1.87 0.45 -4.12 3.86e-05 Observer JLE 0.90 0.06 14.89 < 2e-16

Random Effects Var. SD Maternal Seed Source 0.30 0.55 Soil Microbial Inoculum Source 1.49 1.22 Shadehouse Bench 0.16 0.40

We then extracted the logit-linked coefficient estimates for each observer (x) from this model, then back-transformed the estimates using Equation A1, to obtain estimates of proportion colonization by AM fungi for each observer (prop.AMF).

Table 22: Coefficient estimates for proportion colonization of AM fungi for each observer. Logit-linked coefficient estimates (x) and back-transformed coefficient estimates (prop.AMF).

x prop.AMF Observer CSD -1.87 0.13 Observer JLE -0.96 0.28 152

We then used Equation A2 to calculate the difference in coefficient estimates between the two observers (o), i.e., the average amount that Observer JLE scored above Observer

CSD. This difference (o) was subtracted from all observations by observer JLE to create a standardized variable of proportion colonization by AM fungi correcting for systemic over-scoring by Observer JLE in relation to Observer CSD.

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Table 23: Mixed-model ANCOVA of AMF colonization in offspring vs. non- offspring seedlings based on arbuscules only. ANCOVA table of the predictors of AMF colonization of offspring vs. non-offspring experimental seedlings in conspecific soil microbial communities at the end of the 8-month soil microbial inoculation shadehouse experiment (n = 112 seedlings) based on arbuscules only. Experimental treatment = whether a seedling was the offspring or a non-offspring of the adult tree whose soil microbial community it was grown in. Initial biomass = a seedling’s initial dry biomass (estimated). Observer = which of two researchers quantified AMF colonization in the seedling. Fixed effects summary includes log-transformed coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), z-score (z = β/SE(β)), F-value (F), and significance level (p). The intercept represents seedlings in the offspring treatment. Random effects summary includes variance and standard deviation of all random effects (i.e., 11 maternal seed sources, 11 soil microbial inoculum sources, and 4 shadehouse benches).

Fixed Effects β SE(β) z df F p Intercept -2.63 0.43 -6.08 NA NA 1.23e-09

Non-offspring Seedlings 0.31 0.12 2.52 99.54 1.91 0.01

Initial Biomass 0.84 0.48 1.76 106.54 1.02 0.08

Observer: JLE -3.13 0.21 -15.23 105.73 21.66 < 2e-16

Random Effects Var. SD Maternal Seed Source 0.33 0.58 Soil Microbial Inoculum Source 0.56 0.75 Bench 0.23 0.48

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Appendix B: Soil Nutrients (Chapter 1)

Figure 38: PCA of nutrients in the soil inocula. A scree plot showing that total phosphorous (in red) is the best indicator of overall soil nutrient status in the 11 soil inocula in the experiment of the eight soil nutrient variables tested.

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Appendix C: Conspecific Seedling Density (Chapter 3)

Table 24: Mixed-model ANCOVA of the survival of seedlings in plots with 4 seedlings only in male and female environments in the field. ANCOVA table of the predictors of survival of the V. surinamensis seedlings in plots with four conspecific seedlings only in male vs. female environments at the end of the 7-month field experiment (n = 58 seedlings). Intercept = the probability of survival of unclipped seedlings in female environments; male environment = the impact of being in a male environment on seedling survival; clipping = the impact of clipping on seedling survival; initial biomass = the impact of initial biomass on seedling survival. Fixed effects summary includes logit-linked coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), z-score (t = β/SE(β)), F-value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (n = 6 maternal seed sources, 10 focal adults, and 43 seedling plots).

Fixed Effects β SE(β) df z F p

Intercept -2.40 0.97 NA -2.47 NA 0.01

Male Environment -1.37 0.91 4.25 -1.50 0.49 0.13

Clipping -0.78 0.99 50.34 -0.78 0.29 0.43

Initial Biomass 5.1 2.37 30.73 2.37 7.79 0.02

Random Effects Var. SD Maternal Seed Source 0.00 0.00 Focal Adult 0.00 0.00 Plot 0.23 0.48

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Table 25: Mixed-model ANCOVA of the biomass of seedlings in plots with 4 seedlings only in male and female environments in the field. ANCOVA table of the predictors of biomass of the V. surinamensis seedlings in plots with four conspecific seedlings only in male vs. female environments at the end of the 7-month field experiment (n = 13 seedlings). Intercept = the biomass of unclipped seedlings in female environments; male environment = the impact of being in a male environment on seedling biomass; clipping = the impact of clipping on seedling biomass; initial biomass = the impact of initial biomass on seedling biomass. Fixed effects summary includes coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), t-score (t = β/SE(β)), F-value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (n = 4 maternal seed sources, 6 focal adults, and 9 seedling plots).

Fixed Effects β SE(β) df t F p

Intercept 0.57 0.14 8.88 4.00 NA 3.21e-03

Male Environment -0.04 0.21 6.92 -0.17 0.03 0.87

Clipping -0.27 0.14 4.26 -1.97 3.88 0.12

Initial Biomass 0.12 0.28 4.69 0.43 0.19 0.68

Random Effects Var. SD Maternal Seed Source 0.008 0.089 Focal Adult 0.000 0.000 Plot 0.070 0.264 Residual 0.008 0.087

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Appendix D: Clipping (Chapter 3)

Model of seedling clipping in the field

We analyzed the effects of conspecific adult sex (male vs. female), conspecific seedling density, the interaction between the seedling-adult relationship and conspecific seedling density, and initial seedling biomass on whether a seedling had been clipped at any point during the experiment (yes/no). A generalized linear mixed-effects model with binomial distribution was used to analyze clipping. Maternal seed source, focal tree, and plot were included as random effects in the model.

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Table 26: Mixed-model ANCOVA of clipping in male and female environments in the field. ANCOVA table of the predictors of the probability of clipping of V. surinamensis seedlings in male vs. female environments at the end of the 7-month field experiment (n = 169 seedlings). Intercept = the probability of clipping of seedlings in female environments; male environment = the impact of being in a male environment on clipping; conspecific seedlings = the impact of one additional conspecific seedling neighbor on clipping; initial biomass = the impact of a seedling’s initial biomass on clipping. Fixed effects summary includes coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), z-score (z = β/SE(β)), F-value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (n = 11 maternal seed sources, 10 soil microbial inoculum sources, and 4 shadehouse benches).

Fixed Effects β SE(β) df z F p

Intercept 0.61 2.02 NA 0.30 NA 0.76

Male Environment -0.17 0.73 19.74 -0.23 0.17 0.82

Conspecific 43.56 -0.35 0.42 -0.84 0.78 0.40 Seedlings

Initial Biomass -0.64 1.05 47.19 -0.61 0.38 0.54

Random Effects Var. SD Maternal Seed Source 7.01e-09 8.38e-05 Focal Adult 2.90e-01 5.38e-01 Plot 5.94e-09 7.71e-05

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Appendix E: Offspring Seedlings (Chapter 3)

Table 27: Mixed-model ANCOVA of biomass of non-offspring seedlings only in male and female soil microbial communities in the shadehouse. ANCOVA table of the predictors of total dry biomass of non-offspring V. surinamensis seedlings only in male vs. female soil microbial communities at the end of the 8-month soil microbial inoculation shadehouse experiment (n = 112 seedlings). Intercept = seedlings grown in non-mother female soil microbial communities; Male soil microbial communities = impact of being grown in a male soil microbial community. Initial biomass = a seedling’s estimated biomass at the beginning of the experiment. Fixed effects summary includes coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), t-score (t = β/SE(β)), F-value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (n = 11 maternal seed sources, 10 soil microbial inoculum sources, and 4 shadehouse benches).

Fixed Effects β SE(β) df t F p

Intercept 1.28 4.49e-01 6.30 2.85 NA 0.03

Male soil microbial 0.00 9.95e-03 2.36e-01 7.66 0.04 0.97 community

Initial Biomass 3.64 5.03e-01 1.02e-02 7.24 49.76 8.47e-11

Random Effects Var. SD Maternal Seed Source 0.05 0.23 Soil Microbial Inoculum Source 0.09 0.29 Bench 0.54 0.73 Residual 0.50 0.71

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Table 28: Mixed-model ANCOVA of colonization by AM fungi of non-offspring seedlings only in male and female soil microbial communities in the shadehouse. ANCOVA table of the predictors of proportion colonization by AM fungi of non- offspring V. surinamensis seedlings only in male vs. female soil microbial communities at the end of the 8-month soil microbial inoculation shadehouse experiment (n = 94 seedlings). Intercept = seedlings grown in non-mother female soil microbial communities and quantified by observer 1; male soil microbial communities = seedlings grown in male soil microbial communities; initial biomass = a seedling’s estimated biomass at the beginning of the experiment; observer = seedlings quantified by observer 2. Fixed effects summary includes logit-linked coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), t-score (t = β/SE(β)), F-value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (n = 11 maternal seed sources, 10 soil microbial inoculum sources, and 4 shadehouse benches).

Fixed Effects β SE(β) df t F p

Intercept 0.25 0.08 30.13 3.23 NA 0.003

Male soil microbial 0.07 0.06 8.27 1.18 1.39 0.27 community

Initial Biomass -0.08 0.15 88.01 -0.52 0.27 0.60

Observer 0.16 0.04 86.56 3.96 15.68 1.54e-04

Random Effects Var. SD Maternal Seed Source 0.002 0.044 Soil Microbial Inoculum Source 0.005 0.067 Bench 0.004 0.065 Residual 0.030 0.174

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Table 29: Mixed-model ANCOVA of survival of non-offspring seedlings only in male and female environments in the field. ANCOVA table of the predictors of survival of non-offspring V. surinamensis seedlings only in male vs. female environments at the end of the 7-month field experiment (n = 107 seedlings). Intercept = unclipped seedlings in non-mother female environments; male soil microbial communities = the impact of male environments on seedling survival; number of conspecific seedling neighbors in a plot = the impact of adding one conspecific seedling to a plot on seedling survival; clipping = the impact of clipping on seedling survival; initial biomass = a seedling’s estimated biomass at the beginning of the experiment. Fixed effects summary includes logit-linked coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), z-score (z = β/SE(β)), F-value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (n = 6 maternal seed sources, 10 focal adults, and 43 plots).

Fixed Effects β SE(β) df z F p

Intercept 6.66 5.79 NA 1.15 NA 0.25

Male Environment -2.48 2.85 18.10 -0.87 0.36 0.38

Number of -2.39 Conspecific -3.55 1.49 40.03 4.09 0.02 Seedlings in a Plot

Clipping 0.97 1.21 94.83 0.80 0.19 0.42

Initial Biomass 15.03 6.54 62.02 2.30 12.53 0.02

Random Effects Var. SD Maternal Seed Source 4.76 2.18 Focal Adult 10.74 3.28 Plot 0.00 0.00

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Table 30: Mixed-model ANCOVA of biomass of non-offspring seedlings only in male and female environments in the field. ANCOVA table of the predictors of biomass of non-offspring V. surinamensis seedlings only in male vs. female environments at the end of the 7-month field experiment (n = 19 seedlings). Intercept = unclipped seedlings in non-mother female environments; male soil microbial communities = the impact of male environments on seedling biomass; conspecific seedlings in plot = the impact of adding one conspecific seedling to a plot on seedling biomass; clipping = the impact of clipping on seedling biomass; initial biomass = the impact of a seedling’s estimated biomass at the beginning of the experiment on final biomass. Fixed effects summary includes coefficient estimates (β), standard errors (SE(β)), degrees of freedom (df), t-score (t = β/SE(β)), F-value (F), and significance level (p) for each predictor. Random effects summary includes variance and standard deviation of each random effect in the model (n = 6 maternal seed sources, 10 focal adults, and 43 plots).

Fixed Effects β SE(β) df t F p

Intercept 0.75 0.56 13.04 1.34 NA 0.20

Male Environment -0.06 0.18 13.02 -0.30 0.09 0.77

Conspecific -0.50 -0.06 0.12 13.09 0.25 0.63 Seedlings in Plot

Clipping -0.25 0.03 1.12 -9.94 98.73 0.05

Initial Biomass 0.42 0.08 1.19 5.08 25.76 0.09

Random Effects Var. SD Maternal Seed Source 0.00 0.00 Focal Adult 3.23e-15 5.69e-08 Plot 5.79e-02 2.41e-01 Residual 6.66e-04 2.58e-02

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