Causes and Consequences of Fungal Disease in Cottonwoods

CAUSES AND CONSEQUENCES OF FUNGAL DISEASE IN COTTONWOODS

A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOLOGY AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

Posy Elizabeth Busby March 2012

This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/

This dissertation is online at: http://purl.stanford.edu/nj466rp7444

ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Rodolfo Dirzo, Primary Adviser

I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Christopher Field

I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Peter Vitousek

I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.

George Newcombe

I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Thomas Whitham

Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education

This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives.

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Abstract

Plant pathogens are microorganisms that cause disease and are therefore of great economic and ecological importance. Tremendous effort has been devoted to understanding plant genetic resistance to individual pathogens. Yet we know less about how natural genotypic variation in host plants affects the structure of pathogen communities. Here I test how both plant genotype and the environment influence the structure of fungal leaf pathogen communities, and how pathogens in turn affect plant herbivores. First, I test how the evolutionary history of plants affects their pathogen communities. I show that the pathogens of Populus angustifolia, the narrow leaf cottonwood of the Rocky Mountains, are consistent with a hypothesis of Beringian migration into North America by the ancestor of P. angustifolia. Second, in common garden studies and in the wild, I test how inter- and intraspecific genetic variation in Populus affects the structure of pathogen communities. I found that pathogen communities varied both among Populus species and genotypes within species, mostly in the severities of damage inflicted by constituent pathogens rather than in composition. These results suggest that quantitative genetic resistance, which is neither complete nor pathogen-specific, is affecting these communities. Third, I test how environmental variation affects both pathogen communities, and genetic resistance to pathogens. I found that the influence of plant genotype on pathogen communities attenuates as spatial scale and environmental heterogeneity increase, and that plant genetic resistance to pathogens is correlated to the level of disease risk within the environment where the plant occurs. Finally, I test how plant genotypic variation in pathogen severity influences pathogens’ indirect effects on herbivores. I found that pathogens reduce the richness and abundance of herbivores most strongly on severely infected Populus species. Pathogens also reduced herbivory, but this effect was modulated by genotypic variation in herbivore resistance. Pathogens most strongly deterred herbivores on herbivore-susceptible genotypes, suggesting that they induce defenses. Overall, this thesis demonstrates that the genetic-based interactions of relatively few species (e.g., a plant and a pathogen) can be fundamental to understanding community structure.

Acknowledgements

This thesis was completed with the support and encouragement from mentors, collaborators, friends and family. I thank:

My Advisor Rodolfo Dirzo

My Committee Chris Field Peter Vitousek Thomas Whitham George Newcombe

My Chair Terry Root

Collaborators Charlie Canham, Cathie Aime, David Weston, Naupaka Zimmerman, Jamie Lamit, Art Keith and Roger Guevara

Colleagues at Stanford, Northern Arizona University and the University of Idaho Rachel Adams, Hillary Young, Beth Pringle, Camila Donati, Erin Kurtin, Eric Abelson, Mar Sobral, Ted Raab, Bill Anderegg, Tad Fukami, Matt Knope, Alan Le, Denis Willet, Nicole Sarto, Dave Wilson, Ray Itter, Dahlia Wist, Matt Zinkgraf, Matt Lau, Sharon Ferrier, Faith Walker, Mary Ridout and Anil Raghavendra

My Cohort Camila Donati, Beth Pringle, Julie Stewart, Nishad Jayasundara, Kevin Miklasz, Malin Pinsky, Eben Broadbent, Shelby Sturgis, Henri Folse and Jason Ladner

Stanford University Staff Valeria Kiszka, Jennifer Mason, Matt Pinheiro and Dan King

Funding Stanford Department of Biology Department of Energy Global Change Environmental Program Heinz Foundation Beech Tree Trust and the Coalition for Buzzards Bay Torrey Botanical Society

My Family Tim Warren Rich, Sandy, Sibyl, Gwen and Sunny Busby

Table of Contents

Abstract...... iv Acknowledgements...... v Table of Contents...... vi List of Tables ...... ix List of Figures...... x Introduction...... 1 Statement on multiple authorship ...... 4 References...... 5

Chapter 1: Foliar pathogens of Populus angustifolia are consistent with a hypothesis of Beringian migration into North America...... 8 1.1 Abstract...... 9 1.2 Introduction...... 10 1.3 Materials and methods...... 12 1.3.1 Survey of foliar pathogens of P. angustifolia...... 12 1.3.2 Phylogenetic relationships...... 13 1.3.3 Inoculation experiments...... 15 1.4 Results...... 16 1.4.1 Survey of foliar pathogens of P. angustifolia...... 16 1.4.2 Phylogenetic relationships...... 17 1.4.3 Taxonomy of new species ...... 18 1.4.4 Inoculation experiments...... 19 1.5 Discussion...... 20 1.5.1 Support for a Beringia hypothesis ...... 20 1.5.2 Host shifting and community consequences...... 21 1.6 Acknowledgments ...... 22 1.7 References...... 22 1.8 Tables...... 26 1.9 Figures ...... 28

1.10 Supplementary tables...... 33

Chapter 2: Genetic basis of pathogen community structure in foundation tree species...... 40 2.1 Abstract...... 41 2.2 Introduction...... 42 2.3 Materials and methods...... 44 2.3.1 Study system...... 44 2.3.2 Pathogen community ...... 44 2.3.3 Pathogens communities in a common garden ...... 46 2.3.4 Pathogen communities in the wild...... 47 2.3.5 Statistical analyses...... 48 2.4 Results...... 49 2.4.1 Pathogens communities in a common garden ...... 49 2.4.2 Pathogen communities in the wild...... 50 2.5 Discussion...... 51 2.5.1 Plant species effects on pathogens...... 51 2.5.2 Plant genotype effects on pathogens...... 52 2.6 Acknowledgments ...... 53 2.7 References...... 53 2.8 Tables...... 60 2.9 Figures ...... 63

Chapter 3: Effects of plant genotype on associated pathogen community attenuate as environmental heterogeneity increases ...... 69 3.1 Abstract...... 70 3.2 Introduction...... 71 3.3 Materials and methods...... 73 3.3.1 Study system...... 73 3.3.2 Pathogen community ...... 74 3.3.3 Reciprocal transplant experiment ...... 75

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3.3.4 Reciprocal inoculation experiment ...... 77 3.4 Results...... 78 3.4.1 Reciprocal transplant experiment ...... 78 3.4.2 Reciprocal inoculation experiment ...... 79 3.5 Discussion...... 80 3.5.1 Genotypic effects at the local scale ...... 80 3.5.2 Genotypic effects at the landscape scale ...... 81 3.6 Conclusions...... 83 3.7 Acknowledgments ...... 83 3.8 References...... 83 3.9 Tables...... 89 3.10 Figures ...... 92 3.11 Supplementary Figures ...... 96

Chapter 4: Plant genetic resistance modulates the indirect effects of pathogens on arthropod communities and herbivory in foundation trees ...... 97 4.1 Abstract...... 98 4.2 Introduction...... 99 4.3 Results and discussion ...... 101 4.4 Acknowledgements...... 103 4.5 References...... 103 4.6 Figures ...... 108 4.7 Supplementary materials and methods ...... 111 4.7.1 Tree propagation...... 111 4.7.2 Pathogen inoculation ...... 111 4.7.3 Arthropod and herbivory surveys ...... 112 4.7.4 Plant biomass measurements ...... 113 4.7.5 Statistical analyses...... 113 4.8 Supplementary tables...... 115 4.9 Supplementary figures...... 119

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List of Tables

Table 1.1 Range-wide survey of pathogens of Populus angustifolia Table 1.S1 DNA sequences used in Mycosphaerella phylogenetic analyses Table 2.S2 DNA sequences used in Melampsora phylogenetic analyses

Table 2.1 Plant species, hybrid, and genotype effects on pathogen communities Table 2.2 Plant genotype effects on pathogen communities

Table 2.3 Distribution of pathogens on Populus angustifolia, P. fremontii and their F1 hybrid along the Weber River, Utah Table 3.1 Plant genotype effects on pathogen communities within common gardens Table 3.2 Plant genotype effects on pathogen communities among common gardens Table 3.3 Pathogen effects on Populus angustifolia performance Table 4.S1 Model results for leaf biomass Table 4.S2 Foliar arthropods encountered in surveys Table 4.S3 Model results for arthropod community structure and herbivory Table 4.S4 Model results for herbivore and predator community structure

List of Figures

Figure 1.1 Geographic range of Populus angustifolia Figure 1.2 Microscopic images of the conidia of Mycosphaerella musivoides, M. angustifoliorum and M. wasatchii. Figure 1.3 Frequency of Mycosphaerella spp. along the Weber River Figure 1.4 Mycosphaerella phylogenetic tree based on maximum parsimony analysis Figure 1.5 Melampsora phylogenetic tree based on maximum parsimony analysis Figure 2.1 Photographs of the fungal leaf pathogen community Figure 2.2 NMDS analysis of pathogen communities for genotypes within Populus

angustifolia, P. fremontii and their F1 hybrid in the common garden Figure 2.3 Pathogenic severities for Populus species and their hybrid, and for genotypes within Populus species and their hybrid, in the common garden Figure 2.4 NMDS analysis of pathogen communities for Populus angustifolia, P.

fremontii and their F1 hybrid in the wild Figure 2.5 Relationship between P. angustifolia density and pathogen community structure in the wild Figure 2.6 Pathogenic severities in natural stands along the Weber River Figure 3.1 Temperature and relative humidity in common gardens Figure 3.2 Photographs of the fungal leaf pathogen community Figure 3.3 Pathogenic severities in common gardens Figure 3.4 Elevation gradient, associated disease risk, and their relationship to genetic resistance Figure 3.S1 Pathogenic severities in natural stands along the Weber River Figure 4.1 Inter- and intraspecific variation in resistance phenotypes Figure 4.2 Interspecific variation in arthropod richness and abundance Figure 4.3 Inter- and intraspecific variation in herbivory Figure 4.S1 NMDS analysis of arthropod communities on control and pathogen- inoculated plants

Introduction

Understanding why particular species are found in some communities but not in others is a central aim in ecology. My thesis explores why pathogens – microorganisms that cause disease – are found on some plants but not on others, and the consequences of particular pathogens for associated arthropod communities, and for plant-herbivore interactions. The answers to these questions are relevant to our understanding of the natural world, and also to agriculture and conservation since plant pathogens can be both a destructive force causing mortality in plants but also a force maintaining diversity in plant communities (Russell 1987; Packer & Clay 2000; Rizzo & Garbelotto 2003; Lovett et al. 2006; Busby & Canham 2011). My thesis focuses on how genotypic variation in plants affects the structure of pathogen communities. Previous work has shown that genetic variation in foundation plant species, or those that create locally stable conditions for many associated species (Dayton 1972), can affect communities of soil microbes, arthropods, vertebrates and understory plants (Antonovics 1992; Agrawal 2003; Neuhauser et al. 2003; Whitham et al. 2003; Shuster et al. 2006; Whitham et al. 2006; Johnson & Stinchcombe 2007; Hughes et al. 2008; Adams et al. 2011; Rowntree et al. 2011). This work has been influential in demonstrating the potential for evolution in plants to affect the ecology of diverse groups of interacting species. However, to a large degree pathogens have been overlooked in this literature (but see Barbour et al. 2009). Hundreds of studies have evaluated plant genetic resistance to individual pathogen species in natural and agricultural systems (Gilbert 2002). We know that individual pathogens are strongly influenced by genetic resistance and in turn can impose selection pressure on plant populations (Flor 1955; Burdon & Thrall 1999), and that pathogens can be keystone species defining entire communities and ecosystems (Rizzo & Garbelotto 2003; Lovett et al. 2006; Busby & Canham 2011). Understanding the role of plant genetics in shaping functionally and taxonomically diverse pathogen communities, and their interactions with associated species, is important because the cumulative effects of co-occurring pathogens on plant fitness can be non-additive (Fernando & Watson 1994; Morris et al. 2007).

It is in this context that my thesis asks: 1) How does plant genotype and the environment shape pathogen communities of foundation tree species? 2) Does the influence of plant genotype on pathogen communities depend on the degree of environmental heterogeneity? And 3) Does genotypic variation in plant resistance modulate the indirect effects of pathogens on associated arthropod communities and plant-herbivore interactions? I focus on cottonwoods (Populus angustifolia, P. fremontii and their naturally occurring F1 hybrid P. × hinckleyana), and their foliar fungal pathogens along the Weber River in Utah. Cottonwoods are foundation species of riparian forest ecosystems in the Western US, which are currently listed as a threatened habitat type and a hotspot of biodiversity (Noss et al. 1995). In Chapter 1, I test how the evolutionary history of plants affects their pathogen communities. I evaluated host ranges, host preferences, and phylogenetic relationships for pathogens of Populus angustifolia, the narrow leaf cottonwood of the Rocky Mountains. I found that some pathogens display evidence of Eurasian ancestry, while others have recently diversified from North American pathogen species. Because hosts and their obligate symbionts can display parallel phylogeographic patterns, I argue that this community is consistent with a Beringian migration into North America by the ancestor of P. angustifolia. While it is not known precisely when P. angustifolia migrated into North America, my results suggest that some pathogens migrated with the ancestor of P. angustifolia, while others have likely shifted onto P. angustifolia since its arrival. This work thus raises the question of whether the importance of plant genetics for dependent organisms depends on the nature and duration of the association. I expect that plants will have weaker genetic effects on newly acquired symbionts than ancient ones, although this is a topic that warrants additional research. Chapter 2 tests how inter- and intraspecific genetic variation in Populus influences dependent fungal leaf pathogen communities. Both in a common garden and in natural forests throughout the Weber River drainage system in Utah, I found that interspecific genetic variation affects the local and geographic distribution of leaf pathogens in a similar fashion as other diverse organisms (e.g., arthropods, understory plants, soil microbes). In the common garden, pathogen communities also differed among genotypes within species, demonstrating the potential for evolution in plants to

affect the ecology of pathogen communities. Pathogen communities varied primarily in the severities of constituent pathogens rather than in composition, suggesting the importance of quantitative genetic resistance, which is neither complete nor pathogen- specific, for structuring these communities. The role of different types of plant genetic resistance in shaping pathogen communities is a topic that warrants further research (Poland et al. 2009). In Chapter 3, I test whether the influence of plant genotype on pathogen communities depends on the degree of environmental heterogeneity. In a reciprocal transplant experiment utilizing replicated genotypes of P. angustifolia in three common gardens located along an elevational and distance gradient (55 km), I found that the effect of plant genotype on pathogen communities was stronger locally within common gardens than among gardens. This result is consistent with the findings of Bangert et al. (2008), who demonstrated that genetic influences on community organization attenuate as spatial scale increases. I also found that the influence of plant genotype on pathogens differed in different common gardens (genotype by environment interaction). A reciprocal inoculation greenhouse experiment utilizing a single pathogen species confirmed that environmental setting influences genetic resistance to pathogens: seedlings originating from natural stands characterized by high disease risk were more resistant than seedlings originating from natural stands characterized by low disease risk. These results support the geographic mosaic theory of coevolution (Thompson 2005) by illustrating how an environmental gradient can create a selection pressure gradient, leading to differential genetic resistance. These results should be of conservation interest if rapid climate change shifts environments and associated disease pressure outside of the range to which plants have adapted (Harvell et al. 2002). In Chapter 4, I turn to the ecological consequences of pathogens. Indirect interactions are known to play a major role in structuring ecosystems and food webs (Hairston et al. 1960; Paine 1966; Estes & Palmisano 1974), yet the factors that determine the strength of such interactions are often not known. Here, I test whether inter- and intraspecific genetic variation in plant pathogen and herbivore resistance modulates the indirect effects of pathogens on arthropod communities and herbivory.

I found that pathogen inoculation altered arthropod community composition, reduced the richness and abundance of arthropods by as much as 50%, and reduced herbivory by 50%. Interspecific variation in resistance to the pathogen partially modulated arthropod community effects, with heavily defoliated plants strongly affected. In contrast, intraspecific variation in resistance to herbivores modulated the herbivory effect. Pathogens strongly reduced herbivory on the most herbivore-susceptible genotypes, suggesting that pathogen inoculation induced defenses. Previous studies have shown that pathogen infection can induce a jasmonate release response, which can then negatively affect herbivores (Thaler et al. 2001; 2010). Overall, my study supports previous work finding that pathogens, native and exotic, affecting foundation plants can lead to significant community and ecosystem consequences (Ellison et al. 2005; Lovett et al. 2006; Busby & Canham 2011). I further demonstrate how such community consequences can be modulated by genetic variation in resistance to both pathogens and herbivores.

Statement of multiple authorship

I am the first author and primary contributor (data collection, analysis, and writing) of each of my dissertation chapters. This work has benefited from the collaborative efforts of several colleagues. Chapter 2 was co-authored with M. Cathie Aime (Louisiana State University) and George Newcombe (University of Idaho). George was instrumental in the inception of ideas and development of formal descriptions for two new species of Mycosphaerella. Cathie contributed Melampsora DNA sequence data, conducted the Melampsoara phylogenetic analysis, and provided advice on Mycosphaerella phylogenetic analyses. Chapters 2 and 3 were co-authored with Rodolfo Dirzo (Stanford University), Thomas Whitham (Northern Arizona University) and George Newcombe. Data presented in these chapters was collected in natural stands and common gardens that Tom established along the Weber River in Utah in the 1990s. Tom and Rodolfo were instrumental in the inception of ideas and experimental design for these chapters. George helped with pathogen identification and inoculation experiments. Chapter 4 was co-authored with Jamie Lamit, Art Keith, Tom Whitham, George

Newcombe and Rodolfo Dirzo. Jamie raised the cuttings for the experiment, and helped with data processing and analysis. Art Keith surveyed arthropod communities on the plants. Rodolfo contributed to herbivory measurements. Tom, Rodolfo and George each provided feedback on the experimental design. Co-authors of each chapter have contributed feedback on my writing.

References

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Chapter 1

Foliar pathogens of Populus angustifolia are consistent with a hypothesis of Beringian migration into North America

Posy E. Busbya*, M. Catherine Aimeb and George Newcombec

a Stanford University, Department of Biology, 371 Serra Mall, Stanford CA 94305 USA b Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803 USA c Department of Forest Ecology and Biogeosciences, University of Idaho, Moscow ID 83844-1133 USA

1.1 Abstract

Populus angustifolia, the narrowleaf cottonwood, is considered one of two native species of Populus section Tacamahaca restricted to western North America. Efforts to construct a definitive phylogeny of Populus are complicated by ancient hybridization, but some phylogenetic analyses suggest P. angustifolia is more closely related to Asian congeners than to P. trichocarpa, the other species of Populus section Tacamahaca in western North America. Because hosts and their obligate symbionts can display parallel phylogeographic patterns, we evaluated the possibility of a Beringian migration of an Asian ancestor of P. angustifolia by determining the distributions, host preferences, and, in some cases, closest phylogenetic relatives of fungal leaf pathogens of P. angustifolia. Phyllactinia populi, a common foliar pathogen on Populus in Asia but unknown on P. trichocarpa, was found on P. angustifolia in multiple sites. Mycosphaerella angustifoliorum, also unknown on P. trichocarpa, was commonly collected on P. angustifolia. Conversely, many common foliar pathogens of P. trichocarpa in western North America were not found on P. angustifolia; only Melampsora × columbiana and Drepanopeziza populi were common to both Populus species. Phylogenetic analyses revealed that Mycosphaerella angustifoliorum was not part of the diversification of Mycosphaerella on Populus that includes all other Mycosphaerella species on Populus in North America: M. populicola, M. populorum, M. musivoides sp. nov. and M. wasatchii sp. nov. The latter two species represent a newly discovered diversification of M. populorum in western North America. Cross-inoculation experiments suggest that the two shared pathogens, M. × columbiana and D. populi, have shifted from P. trichocarpa to P. angustifolia. Overall, the leaf pathogen community of P. angustifolia is consistent with a Beringian migration into North America by the ancestor of P. angustifolia.

1.2 Introduction

Biogeographic interchange has played a major role in shaping the flora and fauna of North America. The Isthmus of Panama pushed above sea level in the late Cenozoic and led to the Great American Interchange between North and South America. Likewise, migrations via Beringia during the Neogene (starting 23 Mya) allowed North American plants into Asia and vice versa (Wolfe 1987). For example, extant species of Crataegus in eastern Asia are phylogenetically most closely related to those in western North America, and trans-Beringian migrations have been invoked to explain the association (Graham 1999, Lo et al. 2009). Phylogenetic evidence also suggests that Pinus albicaulis (whitebark pine) evolved from an Asian ancestor (Liston et al. 2007); twelve Eurasian pines are more closely related to P. albicaulis than are any North American pines. One of the twelve Eurasian species, Pinus pumila, still grows in northeastern Asia west of the Bering Sea. Extant willows of Salix subgenus Vetrix in the Northern Rocky Mountain region are also thought to have evolved from migrants that arrived via Neogene Beringia (Wolfe 1987). Adaptation to the climate of Beringia may now restrict the descendants of its migrants to high elevation in western North America (e.g., whitebark pine and some shrub willows). Populus suaveolens s.l., a balsam poplar belonging to section Tacamahaca, is common in Chukotka, in far eastern Russia (Hulten 1937), and there is fossil evidence of its presence in Beringia (Ager 2003, Brubaker et al. 2005, Edwards et al. 2005). However, to date, no one has hypothesized that P. suaveolens s.l. or any other Asian species of Populus might have entered North America via Beringia. Populus angustifolia, the narrowleaf cottonwood, is a dominant tree in montane, riparian ecosystems of western North America. P. angustifolia is light- and moisture- demanding, but tolerant of cold conditions (Braatne et al 1996). It is typically found along the upper course (1500-2400 m) of rivers in the Rocky Mountains (Whitham et al. 1999, Eckenwalder 2010), and at lower elevations with either P. fremontii or P. deltoides and their naturally occurring interspecific hybrids. P. angustifolia has become a model for understanding how foundation tree species structure dependent communities, and drive ecosystem processes (Whitham et al. 2006), yet its biogeographic history, and the implications of this history for its ecology, have not been considered.

Eckenwalder’s (1996) analysis of 76 morphological characters suggested two monophyletic groups of Populus section Tacamahaca: “one consisting of the typical balsam poplars, like P. balsamifera and P. trichocarpa, and the other of three, somewhat anomalous, narrow-leaved and thin-twigged species resembling P. angustifolia.” This result divided the three balsam poplar species native to North America into two clades: P. balsamifera and P. trichocarpa in one clade, and P. angustifolia in the other. Ancient hybridization in Populus complicates efforts to create a definitive phylogeny that would reveal the biogeographical history of the genus (Hamzeh and Dayanandan 2004). However, a phylogenetic hypothesis based on three noncoding regions of cpDNA sequences suggested a sister relationship between P. angustifolia and P. cathayana (Hamzeh and Dayanandan 2004), one of three Asian taxa belonging to P. suaveolens s.l. (Eckenwalder 1996). In contrast, the other two balsam poplars of North America, P. balsamifera and P. trichocarpa, were sisters to each other (Hamzeh and Dayanandan 2004). Maximum likelihood trees, based on partial 5.8S RNA gene, ITS1 and ITS2, and part of the 28S subunit sequences from Populus species, also showed that P. angustifolia was not closely related to the other two North American species of Populus section Tacamahaca (Hamzeh and Dayanandan 2004). Further analysis of AFLP markers similarly showed a close relationship between P. trichocarpa and P. balsamifera, and a divergent P. angustifolia allied with Asian balsam poplars (Cervera et al. 2005).

Together, phylogenetic analyses of Populus suggest the possibility of a Beringian migration of an Asian ancestor for P. angustifolia, but no conclusive inferences can yet be drawn. Hosts and their obligate symbionts, like goby fish and their shrimp partners, can display parallel phylogeographic patterns (Thompson et al. 2005, Criscione et al. 2006). Here, we characterize fungal leaf pathogens of P. angustifolia, and test whether they show greater affinity to Eurasian fungal pathogens of Populus than to those of P. trichocarpa, which would be consistent with the Beringian migration hypothesis. Our approach involved extensive sampling of fungal pathogen communities of P. angustifolia throughout most of its geographic range, testing for host preferences in greenhouse inoculation experiments, and phylogenetic analysis of several pathogen species.

1.3 Materials and methods

1.3.1 Survey of foliar pathogens of P. angustifolia For two consecutive years (2009 and 2010), we sampled fungal pathogens on diseased leaves of Populus angustifolia, as well as other Populus species and hybrids collected opportunistically, along rivers in the Rocky Mountains (Colorado, Idaho, Utah and Wyoming). In scattered study sites, symptomatic leaves (approximately 25 per tree) were collected from 1-10 haphazardly selected trees per Populus taxon, per stand (Table 1). Our sampling was more intensive along the Weber River (Ogden, Utah), near the center of the range of P. angustifolia. In this area, P. angustifolia occurs at upper elevations (1400 – 2300m) and P. fremontii is found at lower elevations (1300 – 1500m).

Both pure species, their F1 hybrid P. × hinckleyana, and backcross hybrids with P. angustifolia, are found in a 13 km long intermediate zone (Eckenwalder 1984). We collected symptomatic leaves (approximately 25 per tree) from ten individuals of each Populus taxon present (P. angustifolia, P. fremontii and P. × hinckleyana) in twenty stands spanning pure stands and the hybrid zone, a gradient of approximately 100 km and 1300 m in elevation along the Weber River (>400 trees sampled in 2009 and in

2010). We distinguished Populus species and their F1 hybrid by leaf morphology. However, leaf morphology does easily distinguish P. angustifolia from its advanced backcross hybrids. As a result, P. angustifolia samples collected in the Weber River hybrid zone and others may include advanced backcross hybrids that are genetically very similar to P. angustifolia. We identified all fungal pathogens found on leaves collected from all study sites. Species-level identification for Mycosphaerella, the most commonly occurring pathogen, required analysis of conidia micro-morphology. This was not feasible for all specimens. We identified Mycosphaerella species for a subset of the Utah leaf samples collected from pure and hybrid zone stands. In total, 16 and 40 Mycosphaerella specimens (from individual pycnidia) were identified from P. fremontii and P. angustifolia in pure stands, respectively. From the Weber hybrid zone, we examined 13 specimens from P. fremontii, 40 from P. × hinckleyana, and 75 from P. angustifolia. This sampling effort

was biased toward P. angustifolia because Mycosphaerella leaf lesions on P. angustifolia sporulated more readily than those on P. fremontii and P. × hinckleyana. Finally, we queried the Systematic Mycology and Microbiology Laboratory Fungal Database (http://nt.ars-grin.gov/fungalDatabases/index.cfm) to determine the global distribution of fungal taxa on Populus hosts.

1.3.2 Phylogenetic relationships Phylogenetic trees were constructed for Mycosphaerella and Melampsora, the two genera that included unknown, and potentially new species. We used DNA sequence data from our collections and those available from the GenBank database (http://www.ncbi.nlm.nih.gov). Supplementary Table 1 (Mycosphaerella) and Supplementary Table 2 (Melampsora) provide information on all specimens and sequences used in this study. All newly generated sequences have been deposited in GenBank (accessions JQ042235-042251). For each specimen, spore tendrils (Mycosphaerella) or uredinia (Melampsora) were removed with sterile forceps and extracted using the UltraClean plant DNA extraction kit (MoBio Laboratories Inc., Solana Beach, California) following manufacturer’s protocols. DNA extractions were diluted in sterile water and amplified in 25 µl reaction volumes with 12.5 µl of PCR Master Mix (Promega Corp., Madison, WI), 1.25 µl of each (forward and reverse) 10 mM primer and 10 µl of diluted DNA template. For Mycosphaerella three loci were sequenced: the small subunit ribosomal RNA gene (SSU), the beta-tubulin gene (BTUB), and the internal transcribed spacer (ITS) ribosomal RNA gene. Amplification of Mycosphaerella SSU was done with primer pairs NS1/NS4 and NS3/NS8 and PCR parameters described by White et al. (1990); the ITS region was amplified with primer pairs ITS1-F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) and PCR parameters described by Gardes and Bruns (1993); BTUB was amplified with BtExtF/BtExtR and BtMycF/BtMycR primer pairs (Feau et al. 2006). We selected the internal transcribed spacer region 2 (ITS-2) and large ribosomal subunit RNA gene (LSU) for amplification of Melampsora with primers Rust2inv and LR6 following parameters described in Aime (2006). PCR products were cleaned with Montage PCR Centrifugal Filter Devices (Millipore Corp., Billerica, MA) according to the

manufacturer’s protocol and sequenced with BigDye Terminator sequencing enzyme v.3.1 (Applied Biosystems, Foster City, CA) in the reaction: 2 µl of diluted BigDye in a

1:3 dilution of BigDye:dilution buffer (400 mM Tris pH8.0, 10 mM MgCl2); 0.3 µl of 10 mM primer; 10–20 ng of cleaned PCR template; and H2O to 5 µl total reaction volume with amplification primers. Sequencing reactions were cleaned by ethanol precipitation and sequenced on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Contiguous sequences were assembled and edited in Sequencher v.4.1.4 (Gene Codes Corp., Ann Arbor, MI). Sequences were confirmed as belonging to Mycosphaerella or Melampsora by BlastN (http://blast.ncbi.nlm.nih.gov). Sequence alignments for Mycosphaerella were conducted in Geneious Pro 5.3.6TM. For Mycosphaerella, we first constructed trees using individual loci (results not shown), and then using concatenated sequences (ITS+BTUB+SSU). Melampsora sequences were aligned by eye within a dataset of previously published Melampsora sequences (Bennett et al. 2011) in Se-Al v2.0a11 (Andrew Rambaut, Dept. Zoology, University of Oxford, U.K.; http://evolve.zoo.ox.ac.uk/). Based on these preliminary analyses (results not shown) a subset of Melampsora sequences was assembled by pruning redundant Melampsora sequences from Salix species and expanding representative species from the clades to which the newly generated sequences belonged. For both Mycosphaerella and Melampsora trees, maximum parsimony (MP) analyses were conducted in PAUP* v4.0b10 (Swofford 2002) as heuristic searches with 1000 random addition replicates and TBR branch swapping. Support for branches was evaluated by bootstrap analysis derived from 1000 MP replicates with 10 random addition replicates each. Maximum likelihood (ML) analyses were conducted in RAxML-HPC2 v. 7.2.7 (Stamatakis 2006) via the CIPRES portal (Miller et al. 2011) using default parameters adjusting for 1000 bootstrapping replicates. Finally, Bayesian analyses were conducted using the MrBayes plugin in Geneious Pro 5.3.6TM (Huelsenbeck and Ronquist 2001). We used the default settings: chain length 1,100,000, sub sampling frequency 200, burn in length 100,000, with a prior of unconstrained branch lengths (10).

1.3.3 Inoculation experiments Independent inoculation experiments were conducted with Melampsora × columbiana and Drepanopeziza populi, both of which were collected on P. angustifolia and P. trichocarpa (or its hybrid P. × generosa). First, to rule out the possibility of cryptic species specialized on each host, an isolate of each fungal taxon from both host taxa was tested for its pathogenicity (i.e., four isolates, each of which was tested on both host taxa). Second, host preference was tested with the isolates of both fungal taxa from P. angustifolia since a pattern of exapted resistance prevails in Populus and makes it possible to infer that the preferred, or more susceptible, host is the native host (Newcombe 1998, 2005). For the Melampsora experiment, 20 and 30 seedling of P. angustifolia and P. × generosa were grown in a controlled greenhouse environment. Half of the plants (from 20 to 40 cm tall) were inoculated with an isolate of M. × columbiana from P. angustifolia from the Poudre River Canyon of Colorado, where neither P. trichocarpa nor P. × generosa occurs, and half with an isolate from P. × generosa from the Clatskanie Valley of Oregon, where no P. angustifolia occurs. Leaves of each plant in positions 4, 5, and 6 were inoculated with urediniospores as described previously (Newcombe et al. 2001). Two weeks post-inoculation, the number of uredinia per cm2 was calculated after counting uredinia with the aid of a dissecting microscope in five, 1-cm2 fields per leaf. For the Drepanopeziza experiment, 280 seedlings of P. × generosa and P. angustifolia were grown in a controlled greenhouse environment. Half of the plants (from 10 to 20 cm tall) were inoculated with field inoculum of D. populi from P. angustifolia from the Weber River Canyon of Utah, where neither P. trichocarpa nor P. × generosa occurs, and half with field inoculum from P. × generosa from Moscow, Idaho, where no P. angustifolia occurs. Field inoculum comprised aqueous spore suspension produced from field-collected leaves with sporulating lesions. Conidia were washed off the leaves into sterile, distilled water via agitation in plastic bags; spore suspension was then sprayed onto the plants until the plants were saturated. The plants were placed in black garbage bags for 12 hrs. Two weeks post-inoculation, the severity of pathogen infection was quantified by the percentage of leaf area damaged by Drepanopeziza.

1.4 Results

1.4.1 Survey of foliar pathogens of P. angustifolia We found five fungal taxa to be common on Populus angustifolia: Drepanopeziza populi, Mycosphaerella angustifoliorum, Mycosphaerella musivoides sp. nov., Mycosphaerella wasatchii sp. nov. and Phyllactinia populi (Table 1). A sixth taxon, Melampsora × columbiana, was collected in quantity on P. angustifolia in one site alone: the Poudre River Canyon, Colorado, in both years of the survey. A single uredinium of this rust fungus was observed on one Idaho specimen in the first year of the survey, but no rust was found on Idaho specimens in the second year. We did not find any evidence of the following fungi on P. angustifolia: Erysiphe adunca (the cause of powdery mildew), Taphrina (leaf blisters), and Venturia (leaf and shoot blight). Erysiphe, Taphrina and Venturia are all common on P. trichocarpa and its hybrid P. × generosa in western North America. Many other fungi, less common on P. trichocarpa and P. × generosa, were also absent from P. angustifolia. Phyllactinia populi was common on Populus angustifolia in Utah, and it was also found on this host in Idaho and Colorado (Table 1). Phyllactinia populi occurred infrequently on P. fremontii and P. × hinckleyana in the Weber River hybrid zone, and was absent on P. fremontii in lower-elevation pure stands. A preference for P. angustifolia in this hybrid system suggests that it is the native host. The only record of Phyllactinia on Populus in North America retrieved from the SMML database was for P. angustifolia in New Mexico (Farr et al. n.d.), so records for P. fremontii and P. × hinckleyana are new. In contrast, Phyllactinia mildew is common for species and hybrids of four of the five sections of Populus in Asia (Farr et al. n.d.). Mycosphaerella isolates were common on P. angustifolia and other Populus species sampled in all areas (Table 1). Both M. angustifoliorum and M. populorum were expected on Populus angustifolia in the range of the latter (Ramaley 1991; Farr et al. n.d.). Three distinct conidial morphologies were observed: septate, uncapped conidia (Fig. 2A), aseptate, capped (Fig. 2B), and aseptate uncapped (Fig. 2C). Septate, uncapped conidia initially suggested that the first morphotype was M. populorum

(anamorph Septoria musiva), but this was not confirmed in subsequent phylogenetic analysis. The second morphotype with aseptate, capped conidia were typical of M. angustifoliorum (Ramaley 1991). The third morphotype, with aseptate, uncapped conidia appeared to be novel for Mycosphaerella on Populus, and this was confirmed in subsequent, phylogenetic analysis. For all three morphotypes we observed leaf lesions only, not stem cankers that are characteristic of M. populorum on Populus in eastern and mid-western North America (Newcombe 1996). In Utah, both M. angustifoliorum and the septate, uncapped morphotype were collected on P. angustifolia, P. fremontii, and P. × hinckleyana (Fig 3). The frequency of M. angustifoliorum was higher on P. angustifolia than on P. fremontii, whereas the frequency of the septate, uncapped morphotype (that appeared to be M. populorum but is now described below as M. musivoides sp. nov.) was higher on P. fremontii (Fig 3). The third morphotype, with aseptate, uncapped conidia (subsequently described, below, as M. wasatchii sp. nov.), was found infrequently on both P. angustifolia and P. × hinckleyana in Utah and Wyoming. The only previous record of M. angustifoliorum on Populus in North America was Ramaley’s type collection on P. angustifolia in Colorado (Farr et al. n.d.), so again, records for P. fremontii and P. × hinckleyana expand the known host range of this species. Members of the rust genus Melampsora were found on P. angustifolia in only one site in both years (the Poudre River Canyon near Fort Collins, Colorado). The Poudre River Canyon population proved to be M. × columbiana in terms of urediniospore morphology (Newcombe et al. 2000), and its LSU sequence placed it in the M. occidentalis clade (Fig. 5). Most of the Poudre River Canyon urediniospores had equatorial smooth spots and, although they varied from 21 to 38 µm long, they averaged 29 µm. In contrast, M. occidentalis urediniospores are evenly echinulate and collections average 38 to 50 µm.

1.4.2 Phylogenetic relationships The Mycosphaerella dataset was alignable (after exclusion of indels) across 3108 bp of which 2153 were constant, 690 were parsimony-informative, and 265 variable but uninformative. The default maximum limit of 100 equally parsimonious trees was

reached with a best tree score of 2936. The strict consensus tree shows that M. angustifoliorum evolved from a diversification of Mycosphaerella on Populus distinct from that of M. populicola (North America), M. populorum (North America), and M. populi (Europe) (Fig 4). Additionally, our tree provides support for two new species, M. musivoides sp. nov. and M. wasatchii sp. nov, that are part of the North American diversification (Fig 4). Bootstrap values supported divergence between eastern M. populorum and M. wasatchii sp. nov. (host P. angustifolia) and M. musivoides sp. nov. (host P. fremontii). Mycosphaerella musivoides sp. nov. and M. wasatchii sp. nov., described below, differed phylogenetically (Fig. 4) from M. populorum. Additionally, their asexual states were characterized by larger pycnidia bearing aseptate and 1- to 4-septate conidia, respectively. M. populorum has smaller pycnidia (average and largest diameters of 80 and 128 µm, respectively) with conidia that are more commonly 3- or 4-septate (Thompson 1941) than those of M. musivoides sp. nov. that are more commonly 1- or 2- septate. M. angustifoliorum produces aseptate conidia like M. wasatchii sp. nov., but its capped conidia (Fig. 2B) were described as a Clypeispora anamorph (Ramaley 1991) unlike the Septoria anamorphs of the other species discussed here. The Melampsora dataset was completely alignable across 1478 bp of which 1311 were constant, 118 characters were parsimony-informative, and 49 were variable but uninformative. The default maximum limit of 100 equally parsimonious trees was reached with a best tree score of 244. The strict consensus tree reveals that the population of M. × columbiana on P. angustifolia near Poudre, Colorado, shares more rDNA similarity with M. occidentalis than with M. medusae, though M. × columbiana is a hybrid of these two taxa (Fig 5, Newcombe et al. 2000).

1.4.3 Taxonomy of new species Mycosphaerella wasatchii P.E. Busby & G. Newc., sp. nov. Etymology: Occurrence in the Wasatch Range of the Rocky Mountains. Ascomata unknown. Conidiomata pycnidial, amphigenous, from light to dark brown, almost entirely immersed, ostiolate, from 100 to 180 µm. in diameter, with a thin

pseudoparenchymatous wall. Conidia aseptate, mostly curved, hyaline, truncate at one end, tapered to a point at the other, 27-48 µm long x 4 µm wide. Habitat: On leaves of Populus angustifolia, and P. × hinckleyana. Specimens examined: Populus angustifolia leaves (BPI 882464-holotype)

Mycosphaerella musivoides P.E. Busby & G. Newc., sp. nov. Etymology: Conidia are very similar to those of Septoria musiva, the anamorph of M. populorum. Ascomata unknown. Conidiomata pycnidial, amphigenous, from light to dark brown, almost entirely immersed, ostiolate, from 100 to 180 µm. in diameter, with a thin pseudoparenchymatous wall. Conidia from 1- to 4-septate, although conidia of most individual pycnidia are primarily 1- or primarily 2-septate. Conidia mostly curved, hyaline, truncate at one end, tapered to a point at the other, 27-60 µm long x 3-3.5 µm wide. Habitat: On leaves of Populus angustifolia, P. × hinckleyana and P. fremontii. Specimens examined: Populus fremontii leaves (BPI 882465-holotype)

1.4.4 Inoculation experiments In the Melampsora experiment, seedlings of both P. angustifolia and P. × generosa were successfully infected with isolates of M. × columbiana from each host, confirming pathogen-sharing. The isolate from P. angustifolia preferred P. × generosa over P. angustifolia as the respective uredinial density means were 33.9 and 9.7 uredinia per cm2 (two-sample t-test: t = 9.2, df = 373, p < .001), suggesting that the native host was P. × generosa. In the Drepanopeziza experiment, seedlings of both P. angustifolia and P. × generosa were successfully infected with field inocula from both host sources, confirming pathogen-sharing for this fungus as well. With inoculum from P. angustifolia, lesions were visible 10 and 14 days post-inoculation on P. × generosa and P. angustifolia, respectively. Unexpectedly, premature leaf abscission resulted from inoculation, and this made it impossible to determine host preference in terms of lesion area. Still, it was noted that P. × generosa lost its leaves sooner than P. angustifolia.

This finding, along with the earlier appearance of lesions on the former, indicates that P. × generosa is a more susceptible host than P. angustifolia for this pathogen as well as for Melampsora × columbiana.

1.5 Discussion

1.5.1 Support for a Beringian migration hypothesis In contrast to P. trichocarpa, the leaf pathogen community of P. angustifolia had never been studied across its range in any systematic way. These differences are reflected in the numbers of Systematic Mycology and Microbiology Laboratory Fungal Database fungus-host combinations reported for P. trichocarpa and P. angustifolia: 588 and 49, respectively (Farr et al. n.d.). The leaf pathogen communities of P. trichocarpa and its hybrids with P. deltoides (P. × generosa) are commonly comprised of Melampsora, Mycosphaerella (Septoria anamorphs), Venturia (Pollaccia anamorphs), Drepanopeziza (Marssonina anamorphs), and Taphrina in western North America (Spiers 1984, Newcombe 1996, Newcombe and Bradshaw 1996, Newcombe 2003, 2005, Feau et al. 2006). Powdery mildew on P. trichocarpa and its hybrids is caused by Erysiphe adunca (Braun and Takamatsu 2000, Farr et al. n.d.) and is seen late in the fall at non-damaging levels. Our results reveal that the foliar pathogen community of Populus angustifolia is unlike that of P. trichocarpa. Phyllactinia populi is common in Asia on Populus but in North America it appears to be restricted to the geographic range of P. angustifolia. Mycosphaerella angustifoliorum is similarly restricted to the range of P. angustifolia, and it clearly was not part of the diversification of Mycosphaerella on Populus in North America (Fig. 4). Many pathogens common to P. trichocarpa (e.g., Mycosphaerella populicola, Melampsora occidentalis, Venturia inopina, Taphrina sp.) were absent on P. angustifolia. The two pathogens found on both P. angustifolia and P. trichocarpa, Melampsora × columbiana and D. populi, were both more damaging on P. trichocarpa, suggesting that it is the native host (Newcombe 1998). In fact, the only population of M. × columbiana found on P. angustifolia was phylogenetically indistinguishable from Melampsora occidentalis on P. trichocarpa. If the ancestor of P. angustifolia migrated

into North America via Beringia some pathogens may have co-migrated while others were left behind. Rust fungi in particular might have been left behind given the requirement of heteroecious Melampsora for an alternate host. The shift of a North American Melampsora to P. angustifolia would thus have been to an open niche. Overall, our results are consistent with a Beringian migration into North America by the ancestor of P. angustifolia. This migration could have occurred during one of several periods when continuous land was available for plant migrations: >3 Mya, 60-25 Kya, or 20-18 Kya (Graham 1999). Some of the pathogens we observed may have migrated with the ancestor of P. angustifolia (e.g. P. populi, M. angustifoliorium), whereas others may have shifted onto, or diversified on, P. angustifolia since its arrival into North America (e.g. D. populi, M. × columbiana and M. wasatchii). Our future efforts will coordinate phylogeographic analysis of both Populus spp. and their fungal symbionts to explicitly test the Beringian migration hypothesis, and determine the timing of such a migration.

1.5.2 Host shifting and community consequences As the foundation species for montane, riparian communities in western North America, P. angustifolia has become a model in the field of community genetics, with many studies demonstrating its strong genetic effects on associated communities (e.g., arthropods, birds, and microbes) (Whitham et al. 2006). Whitham and colleagues have shown that natural hybridization between two cottonwood species can facilitate parasite shifting from the evolutionary native host to the nonnative host (Floate and Whitham 1993). We too found evidence for host shifting facilitated by hybridization. In Utah, both Phyllactinia populi and M. angustifoliorum appear to have shifted from P. angustifolia via P. × hinckleyana to P. fremontii. In contrast, M. wasatchii and M. musivoides, may have shifted in reverse from P. fremontii via P. × hinckleyana to P. angustifolia. In shifting, preference may remain for an undetermined period with the native host. While we have observed host shifting and plant genetic effects on entire pathogen communities in P. angustifolia hybrid systems (Chapter 2, 3), our results raise the question of whether the importance of plant genotypic variation for dependent organisms

depends on the nature and duration of the association. Overcoming exapted resistance (Newcombe 1998) to shift to a novel Populus hosts is a process that is likely favored by evolutionary timescales. If P. angustifolia migrated into North America in recent evolutionary history, we would expect less host shifting in P. angustifolia hybrid systems since pathogens have had less time to overcome exapted resistance as they have in older North American Populus hybrid systems. More broadly, we hypothesize that plants will have weaker genetic effects on newly acquired symbionts than they have on ancient ones. P. angustifolia hybrid systems thus provide excellent opportunities for determining how different biogeographical and evolutionary histories affect ecological interactions with dependent organisms.

1.6 Acknowledgements

We would like to thank Amy Rossman, Matthew Knope, Tom Whitham, and Rodolfo Dirzo for comments on an earlier draft of this manuscript. Linley Dixon contributed to sequencing. Denis Willet and Alan Le provided valuable assistance in the field. Mary Ridout, Xiaoming Jia, and Anil Raghavendra provided assistance with the inoculation studies. We are grateful to those who contributed to sampling: Wendy Velman and Vince Guyer at the Bureau of Land Management, Faith Walker, Sunny Busby, Daniel O’Brien, Page Lindsey, and Bill Jacobi. This study was supported by the Department of Energy Graduate Research Environmental Fellowship Program (PEB), Stanford University Field Studies Program (PEB), the Heinz Environmental Fellowship (PEB), and the Louisiana Board of Regents and USDA-APHIS (MCA).

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Newcombe, G., B. Stirling, and H. D. Bradshaw Jr. 2001. Abundant pathogenic variation in the new hybrid rust Melampsora x columbiana on hybrid poplar. Phytopathology 91:981-985. Newcombe, G., B. Stirling, S. McDonald, and H. D. Bradshaw. 2000. Melampsora x columbiana, a natural hybrid of M. medusae and M. occidentalis. Mycological Research 104:261-274. Ramaley, A. W. 1991. Clypeispora and its Mycosphaerella teleomorph. Mycotaxon (USA) 40:13-22. Spiers, A. 1984. Comparative studies of host specificity and symptoms exhibited by poplars infected with Marssonina brunnea, Marssonina castagnei and Marssonina populi. European Journal of Forest Pathology 14:202-218. Thompson, G. E. 1941. Leaf-spot diseases of poplars caused by Septoria musiva and S. populicola. Phytopathology 31:241-254. Thompson, A.R., C.E. Thacker, and E.Y. Shaw. 2005. Phylogeography of marine mutualists: parallel patterns of genetic structure between obligate goby and shrimp partners. Molecular Ecology 14:3557-3572 Whitham, T. G., J. K. Bailey, J. A. Schweitzer, S. M. Shuster, R. K. Bangert, C. J. LeRoy, E. Lonsdorf, G. J. Allan, S. P. DiFazio, B. M. Potts, D. G. Fischer, C. A. Gehring, R. L. Lindroth, J. Marks, S. C. Hart, G. M. Wimp, and S. C. Wooley. 2006. A framework for community and ecosystem genetics: From genes to ecosystems. Nature Reviews Genetics 7:510-523. Whitham, T. G., G. D. Martinsen, P. Keim, K. D. Floate, H. S. Dungey, and B. M. Potts. 1999. Plant hybrid zones affect biodiversity: tools for a genetic-based understanding of community structure. Ecology 80:416-428. Wolfe, J. A. 1987. An overview of the origins of the modern vegetation and flora of the northern Rocky Mountains. Annals of the Missouri Botanical Garden 74:785-803.

1.8 Tables

Table 1.1 Range-wide survey of pathogens of P. angustifolia and its hybrids: P. × acuminata [P. deltoides × P. angustifolia] and P. × hinckleyana [P. fremontii × P. angustifolia]. Collections from each site were biased toward diseased leaves [+, ++, and +++ correspond to light, medium and heavy disease severity, respectively]. Where other species of Populus were present, their diseased leaves were also surveyed. Blank cells represent absence of pathogen at the site.

Site [year of Populus Collector Marssonina Mycosphaerella Phyllactinia Melampsora x sampling] taxon populi spp. populi columbiana Weber River angustifolia P. Busby ++ +++ ++ UT [2009] Weber River angustifolia P. Busby +++ ++ ++ UT [2010] Weber River hinckleyana P. Busby + + + UT [2009] Weber River hinckleyana P. Busby + + + UT [2010] Weber River fremontii P. Busby + + + UT [2009] Weber River fremontii P. Busby + + + UT [2010] Dolores River angustifolia Walker ++ +++ CO [2009] San Miguel fremontii Walker + + River CO [2009] San Miguel hinckleyana Walker + + hybrid zone [2009] Red Canyon angustifolia S. Busby ++ near Lander WY [2009] Leadville CO angustifolia O’Brien [2009] Animas River angustifolia Lindsey CO [2009] Animas River deltoides Lindsey CO [2009] San Juan River deltoides Lindsey NM [2009] Animas River acuminata Lindsey CO [2009] San Juan River acuminata Lindsey NM [2009] Kelly Island angustifolia Velman ++ ++ Campground ID [2009] Lorenzo Boat angustifolia Velman +++ +++ + Ramp ID [2009] Heise Bridge angustifolia Velman ++ +++ ID [2009] Wolf Flats ID angustifolia Velman ++ +++ +

[2009] Byington ID angustifolia Velman ++ + [2009] Conant ID angustifolia Velman + +++ + [single [2009] uredinium] Cress Creek angustifolia Velman ++ ++ ++ ID [2009] Annis Island angustifolia Velman + ID [2010] Mud Creek ID angustifolia Velman + [2010] Rattlesnake angustifolia Velman + + Point ID [2010] Poudre River angustifolia Jacobi ++ + + CO [2009] Poudre River acuminata Jacobi + CO [2009] Poudre River deltoides Jacobi CO [2009] Poudre River angustifolia Jacobi ++ CO [2010]

1.9 Figures

Fig 1.1 Geographic range of Populus angustifolia in North America, from Little (1976). http://esp.cr.usgs.gov/data/atlas/little/

Fig 1.2 A) Septate, capless conidia of M. musivoides sp. nov. B) Aseptate, capped conidia of M. angustifoliorum. C) Aseptate, capless conidia of M. wasatchii.

Fig 1.3 Frequency of M. musivoides and M. angustifoliorum on Populus angustifolia, P. fremontii and their F1 hybrid host P. × hinckleyana along the Weber River in Utah. Sample size (i.e., number of pycnidia) for each group is in parentheses. We found only three specimens of M. wasatchii in this survey, two on P. angustifolia and one on P. × hinckleyana.

Fig 1.4 Maximum parsimony trees based on the small subunit ribosomal RNA gene (SSU), the beta-tubulin gene (BTUB), and the internal transcribed spacer (ITS) ribosomal RNA gene for Mycosphaerella spp. Origins of sequences are provided in Supp Table S1. Tree is midpoint rooted. Numbers above branches indicate bootstrapping support (1000 replicates) for each node as ML/MP/Bayesian posterior probability (for those >50). Specimens collected from P. angustifolia, P. fremontii and P. × hinckleyana are in grey.

Fig 1.5 First of 100 equally parsimonious trees based on MP analyses of ITS-2 and LSU sequences of Melampsora spp. Origins of sequences are provided in Supp Table S2. Tree is midpoint rooted. Numbers above branches indicate bootstrapping support (1000 replicates) for each node as ML/MP/Bayesian posterior probability (for those >50). The specimen collected from P. angustifolia is in grey.

1.10 Supplementary tables

Table 1.S1 Sequences used in Mycosphaerella phylogenetic analyses.

TELEOMORPH ANAMORPH LOCALITY HOST GENBANK NO. (ITS, ß-TUBULIN, SSU) ORIGIN

M. populorum S. musiva Canada P. deltoides AY549465 DQ026394 DQ028562 Feau 2006

M. populorum S. musiva Canada P. deltoides AY549464 DQ026396 DQ028564 Feau 2005

M. populorum S. musiva WI, USA NM6 (NxM) DQ029123 DQ026398 DQ028566 Feau 2005

M. populorum S. musiva WI, USA MWH15 (DxM) DQ029124 DQ026397 DQ028565 Feau 2005

M. populorum S. musiva MI, USA Populus hybrid AY549467 DQ026395 DQ028563 Feau 2005

Unknown S. wasatchii Utah, USA P. angustifolia this paper

Unknown S. wasatchii WY, USA P. angustifolia this paper

Unknown S. musivoides Utah, USA P. x hinckleyana this paper

Unknown S. musivoides Utah, USA P. fremontii this paper

M. populicola S. populicola WA, USA P. trichocarpa AY549478 DQ026386 DQ028554 Feau 2005

M. populicola S. populicola DQ019399 DQ026385 DQ028553 Feau 2006

M. populicola S. populicola Canada P. balsamifera DQ019358 DQ026335 DQ028503 Feau 2006

M. populi S. populi France P. nigra DQ029120 DQ026392 DQ028560 Feau 2006

M. populi S. populi Croatia P. nigra DQ029121 DQ026393 DQ028561 Feau 2006

Unknown S. ostryae Canada Corylus cornuta DQ019396 DQ026367 DQ028535 Feau 2006

Unknown S. betulae WI, USA Betula sp. DQ019400 DQ026379 DQ028547 Feau 2006

Unknown S. betulae DQ019376 DQ026378 DQ028546 Feau 2006

Unknown S. betulae Canada Betula populifolia DQ019374 DQ026375 DQ028543 Feau 2006

Unknown S. alnifolia Canada Alnus incana DQ019363 DQ026340 DQ028508 Feau 2005

Unknown S. alnifolia WA, USA Alnus rubra DQ019365 DQ026342 DQ028510 Feau 2006

Unknown S. alnii Canada Alnus incana DQ019364 DQ026341 DQ028509 Feau 2006

M. rubi S. rubi Canada Rubus sp. DQ019369 DQ026346 DQ028514 Feau 2006

M. ribis S. ribis Canada Ribes sp. DQ019367 DQ026344 DQ028512 Feau 2006

M. ribis S. ribis Canada Ribes sp. DQ019368 DQ026345 DQ028513 Feau 2006

Unknown S. canadensis Canada Cornus canadensis DQ019383 DQ026357 DQ028525 Feau 2006

Unknown S. canadensis Canada Cornus canadensis DQ019384 DQ026358 DQ028526 Feau 2006

Unknown S. azaleae Belgium Unknown DQ019366 DQ026343 DQ028511 Feau 2006

Unknown S. floridae DQ019390 DQ026364 DQ028532 Feau 2006

Unknown S. floridae DQ019389 DQ026363 DQ028531 Feau 2006

Unknown S. floridae MI, USA DQ019388 DQ026362 DQ028530 Feau 2006

Unknown S. cornicola Canada Cornus stolonifera DQ019387 DQ026361 DQ028529 Feau 2006

Unknown S. cornicola DQ019386 DQ026360 DQ028528 Feau 2006

Unknown S. cornicola Canada Cornus stolonifera DQ019385 DQ026359 DQ028527 Feau 2006

M. angustifoliorum Clypeispora Utah, USA P. angustifolia this paper

M. brassiciola Asteromella Canada Brassica sp. DQ019330 DQ026313 DQ028481 Feau 2006

M. ulmi S. ulmi Australia Ulmus sp. DQ019377 DQ026370 DQ028538 Feau 2006

M. ulmi S. ulmi DQ019378 DQ026371 DQ028539 Feau 2006

M. latebrosa S. aceris DQ019348 DQ026350 DQ028518 Feau 2006

M. latebrosa S. aceris Canada Acer saccharum DQ019347 DQ026349 DQ028517 Feau 2006

M. latebrosa S. aceris OR, USA Acer circinatum DQ019351 DQ026329 DQ028497 Feau 2006

M. latebrosa S. quercicola Austria Quercus petrea DQ029125 DQ026401 DQ028569 Feau 2006

M. latebrosa S. aceris Canada Acer rubrum DQ019349 DQ026351 DQ028519 Feau 2006

Unknown S. sambucina Canada Sambucus DQ019398 DQ026368 DQ028536 Feau 2006

Unknown S. lycopersicii WI, USA Lycopersicon DQ019395 DQ026366 DQ028534 Feau 2006

Unknown S. helianthi WI, USA Helianthus sp. DQ019394 DQ026365 DQ028533 Feau 2006

Unknown S. glycines Unknown Glycines max DQ019391 DQ026382 DQ028550 Feau 2006

Unknown S. glycines Glycines max DQ019392 DQ026383 DQ028551 Feau 2005

Unknown Cercospora beticola Belgium Beta vulgaris DQ019359 DQ026336 DQ028504 Feau 2006

M. colombiensis Pseudocercospora France Eucalyptus sp. DQ019332 DQ026315 DQ028483 Feau 2006

M. colombiensis Pseudocercospora DQ019331 DQ026314 DQ028482 Feau 2006

M. musicola Pseudocercospora Cameroun Musa sp. DQ019355 DQ026332 DQ028500 Feau 2006

M. musicola Pseudocercospora Colombia Musa sp. DQ019356 DQ026333 DQ028501 Feau 2006

M. musicola Pseudocercospora Indonesia Musa sp. DQ019357 DQ026334 DQ028502 Feau 2006

M. fijiensis Pseudocercospora Philippines Musa sp. DQ019340 DQ026323 DQ028491 Feau 2006

M. fijiensis Pseudocercospora DQ019339 DQ026322 DQ028490 Feau 2006

M. eumusae Pseudocercospora Malaysia Musa sp. DQ019336 DQ026319 DQ028487 Feau 2006

M. eumusae Pseudocercospora DQ019338 DQ026321 DQ028489 Feau 2006

M. eumusae Pseudocercospora DQ019337 DQ026320 DQ028488 Feau 2006

M. laricina Pseudocercospora Switzerland Larix decidua DQ019345 DQ026328 DQ028496 Feau 2006

M. laricina Pseudocercospora DQ019344 DQ026327 DQ028495 Feau 2006

M. laricina Pseudocercospora DQ019343 DQ026326 DQ028494 Feau 2006

M. laricina Pseudocercospora DQ019342 DQ026325 DQ028493 Feau 2006

M. musae unknown Cook Islands Musa sp. DQ019354 DQ026331 DQ028499 Feau 2006

M. musae unknown Cameroun Musa sp. DQ019353 DQ026330 DQ028498 Feau 2006

Unknown S. albopunctata GA, USA Vaccinium DQ019360 DQ026337 DQ028505 Feau 2006

Unknown S. albopunctata DQ019362 DQ026339 DQ028507 Feau 2006

Unknown S. albopunctata DQ019361 DQ026338 DQ028506 Feau 2006

M. dearnessi Lecanosticta acicola France Pinus pinaster DQ019333 DQ026316 DQ028484 Feau 2006

M. dearnessi Lecanosticta acicola DQ019335 DQ026318 DQ028486 Feau 2006

M. graminicola S. triticci France unknown DQ019341 DQ026324 DQ028492 Feau 2006

Botryosphaeria unknown Italy Quercus cerris AF383949 DQ026404 DQ028572 Feau 2006

Unknown S. pini-thunbergii Japan Pinus thunbergii DQ019397 DQ026403 DQ028571 Feau 2006

Feau, N., R. C. Hamelin, and L. Bernier. 2006. Attributes and congruence of three molecular data sets: Inferring phylogenies among Septoria-related species from woody perennial plants. Molecular Phylogenetics and Evolution 40:808-829. Feau, N., J. E. Weiland, G. R. Stanosz, and L. Bernier. 2005. Specific and sensitive PCR- based detection of Septoria musiva, S. populicola and S. populi the causes of leaf spot and stem canker on poplars. Mycological research 109:1015-1028.

Table 1.S2 Origin of Melampsora sequences used in phylogenetic analyses.

TELEOMORPH LOCALITY HOST GENBANK NO. ORIGIN

Melampsora abietis-canadensis Canada FJ666513 Vialle 2009

Melampsora abietis-populi S. Imai China Populus wilsonii AB116799 Tian 2004

Melampsora aecidioides (DC.) J. Schröt. Canada FJ666519 Vialle 2009

“ Canada FJ666510 Vialle 2009

Melampsora allii-populina Kleb. China Populus laurifolia AB116800 Tian 2004

“ China Populus laurifolia AB116801 Tian 2004

“ China Populus talassica AB116802 Tian 2004

“ China Populus laurifolia AB116803 Tian 2004

Melampsora hypericorum (DC.) J. Schröt. Hypericum calycinum AF426196 Maier 2003

Melampsora euphorbiae Castagne Syria Euphorbia macrocladaDQ437504 Aime 2006

Melampsora helioscopiae (Pers.) G. Winter Euphorbia helioscopiaAF426197 Maier 2003

Melampsora laricis R. Hartig China Populus davidiana AB116809 Tian 2004

“ China Populus adenopoda AB116807 Tian 2004

“ China Populus davidiana AB116805 Tian 2004

“ China Populus adenopoda AB116808 Tian 2004

“ China Populus adenopoda AB116819 Tian 2004

Melampsora laricis-pentandrae Kleb. Salix pentandra AY444783 Pei 2005

Melampsora laricis-populina Kleb. CA, USA P. deltoids × nigra JQ042250 this study

“ China Populus simonii AB116772 Tian 2004

“ China Populus nigra AB116784 Tian 2004

“ China Populus purdomii AB116779 Tian 2004

“ China Populus simonii AB116781 Tian 2004

“ Argentina Populus nigra JQ042251 this study

“ China Populus berolinensis AB116786 Tian 2004

“ China Populus cathayana AB116769 Tian 2004

“ China Populus laurifolia AB116788 Tian 2004

“ China Populus simonii AB116785 Tian 2004

“ China Populus popularis AB116770 Tian 2004

“ China Populus nigra AB116783 Tian 2004

“ China Populus canadensis AB116778 Tian 2004

“ China Populus simonii AB116775 Tian 2004

“ China Populus opera AB116774 Tian 2004

“ China Populus simonii AB116782 Tian 2004

“ China Populus nigra AB116771 Tian 2004

“ China Populus maximoniczii AB116776 Tian 2004

“ China Populus cathayana AB116773 Tian 2004

“ P. deltoides × nigra AY444785 Pei 2005

“ P. deltoides × tri. AY444784 Pei 2005

Melampsora medusa Thüm. MN, USA Populus deltoides EF192208 Bennet 2011

“ MN, USA Populus balsamifera JQ042242 this study

“ ND, USA Populus deltoides JQ042243 this study

“ ND, USA Populus deltoides JQ042244 this study

“ ND, USA Populus hybrid JQ042245 this study

“ NY, USA Populus sp. JQ042246 this study

“ LA, USA Populus deltoides JQ042247 this study

Melampsora medusae f.sp. tremuloidis Shain Canada FJ666514 Vialle 2009

Melampsora nujiangensis China Populus yunnanensis AB116820 Tian 2004

Melampsora occidentalis H.S. Jacks. WA, USA Populus balsamifera JQ042237 this study

“ ID, USA Populus trichocarpa JQ042236 this study

“ AF522173 Szaro & Bruns

“ CA, USA Populus trichocarpa JQ042239 this study

Melampsora populnea (Pers.) P. Karst. Populus alba AY444786 Pei 2005

Melampsora populnea f.sp. laricis Boerema & Verh. France FJ666509 Vialle 2009

Melampsora populnea f.sp. pinitorqua Boerema & Verh. FJ666523 Vialle 2009

Melampsora pruinosae Tranzschel China Populus euphratica AB116796 Tian 2004

“ China Populus euphratica AB116795 Tian 2004

“ China Populus euphratica AB116792 Tian 2004

“ China Populus euphratica AB116794 Tian 2004

“ China Populus euphratica AB116793 Tian 2004

Melampsora ribesii-purpureae Kleb. Salix purpurea AY444791 Pei 2005

Melampsora × columbiana CO, USA Populus angustifolia JQ042235 this study

Melampsora sp. USA Populus tremuloides EF192207 Bennett 2011

“ ID, USA Salix amygdaloides EF192206 Bennett 2011

“ ID, USA Populus tremuloides JQ042240 this study

“ CA, USA Populus trichocarpa JQ042241 this study

“ USA Populus tremuloides EF192210 Bennett 2011

“ ID, USA Populus trichocarpa JQ042238 this study

“ USA Populus tremuloides EF192209 Bennett 2011

“ USA Populus tremuloides JQ042248 this study

“ ID, USA Populus tremuloides JQ042249 this study

“ China Populus tremula AB116790 Tian 2004

“ China Populus tomentosa AB116791 Tian 2004

“ China P. alba AB116813 Tian 2004

“ China P. alba AB116815 Tian 2004

“ AZ, USA Salix arizonica EF192193 Bennett 2011

“ ID, USA Salix melanopsis EF192194 Bennett 2011

“ ID, USA Salix bebbiana EF192197 Bennett 2011

“ China Populus yunnanensis AB116821 Tian 2004.

“ AY512866 Begerow unpublished

“ China P. pseudoglauca AB116798 Tian 2004

“ China P. pseudoglauca AB116797 Tian 2004 Aime M.C., Matheny P.B., Henk D.A., Frieders E.M., Nilsson R.H., Piepenbring M., et al. An overview of the higher level classification of Pucciniomycotina based on combined analyses of nuclear large and small subunit rDNA sequences. Mycologia 98:896-905. Bennett C., Aime M.C., Newcombe G. 2011. Molecular and pathogenic variation within Melampsora on Salix in western North America reveal numerous cryptic species. Mycologia 103: 1004-1018. Maier W., Begerow D., Weiss M., Oberwinkler F. 2003. Phylogeny of the rust fungi: an approach using nuclear large subunit ribosomal DNA sequences. Canadian Journal of Botany 81:12-23. Pei M.H., Bayon C., Ruiz C. 2005. Phylogenetic relationships in some Melampsora rusts on Salicaceae assessed using rDNA sequence information. Mycological Research 109:401-409. Tian C., Shang Y., Zhuang J., Wang Q., Kakishima M. 2004. Morphological and molecular phylogenetic analysis of Melampsora species on poplars in China. Mycoscience 45:56-66.

Vialle A., Feau N., Didukh M., Allaire M., Martin F., Moncalvo J.M., Hamelin R.C. 2009. Evaluation of mitochondrial genes as DNA barcode for Basidiomycota. Molecular Ecology Resources 9:99-113.

Chapter 2

Genetic basis of pathogen community structure in foundation tree species

Posy E. Busby,1* Rodolfo Dirzo1, George Newcombe2 and Thomas G. Whitham3

1Stanford University, Department of Biology, 371 Serra Mall, Stanford CA 94305 USA 2College of Natural Resources, University of Idaho, Moscow ID 83844-1133 USA 3Department of Biological Sciences, Northern Arizona University, Flagstaff AZ 86011 USA

2.1 Abstract

Previous studies have shown that genotypic variation in foundation plant species affects arthropod, plant, and soil microbial communities, but less is known about plant genetic relationships with pathogen communities. Moreover, while those studies have revealed important insights, it has been argued that plant genetic effects on associated communities are unrealistically emphasized in common gardens where possible environmental effects are minimized. To critically evaluate these issues, in a common garden and in natural forests throughout the Weber River drainage system (Utah, USA), we test how inter- and intraspecific genetic variation in foundation tree species (Populus angustifolia, P. fremontii and their naturally occurring F1 hybrid P. × hinckleyana) affects fungal leaf pathogen communities (five species from three Orders). In the common garden, pathogen communities differed among both Populus species and their hybrid, and among genotypes within species and their hybrid. In the wild, pathogen communities also differed among both Populus species and their hybrid, but were strongly influenced by the local density of the preferred Populus host species too. Pathogen communities varied mostly in the severities of constituent pathogens rather than in composition, suggesting that quantitative genetic resistance, which is neither complete nor pathogen- specific, is affecting these communities. Overall, the considerable agreement of our findings in the wild and in the common garden suggests that plant genetics can affect the local and geographic distribution of pathogens in a similar fashion as other diverse organisms, and that studies in common gardens can scale to whole watersheds.

2.2 Introduction

Understanding the factors shaping ecological communities is a major objective in biology. The field of community genetics seeks to determine how genotypic variation in one species influences the structure (i.e., species abundances and composition) of associated communities (Antonovics 1992; Agrawal 2003; Neuhauser et al. 2003; Whitham et al. 2003; Shuster et al. 2006; Whitham et al. 2006; Johnson & Stinchcombe 2007; Hughes et al. 2008; Rowntree et al. 2011). In many common garden studies, genetic variation in foundation plant species – those that create locally stable conditions for many associated species (Dayton 1972) – has been shown to influence foliar arthropod communities (Fritz 1988; Dungey et al. 2000; Van Zandt & Agrawal 2004; Wimp et al. 2005; Barbour et al. 2009; Keith et al. 2010). Less is known about how genotypic variation in foundation plant species affects the structure of microbial communities, particularly pathogens. In addition, it has been argued that plant genetic effects are exaggerated in common gardens where environmental effects are largely minimized, but swamped in the wild by environmental variation (Johnson & Agrawal 2005; Tack et al. 2010). To critically evaluate both of these issues, we test for plant genetic effects on pathogen communities in a common garden, and in the wild to determine whether genetic effects scale to a whole watershed. Plant pathogens are microorganisms that cause disease and are therefore of great economic and ecological importance. Pathogens can devastate plant populations by causing reduced fitness and mortality (Gilbert 2002). Yet pathogens can also enhance plant species diversity within communities (Webb 1999; Harms et al. 2000; Packer & Clay 2000) and genetic diversity within host populations (Haldane 1949; Jarosz & Levy 1988). For these reasons, considerable research effort has been devoted to understanding how the plant immune system has evolved to resist individual, often damaging pathogens (Jones & Dangl 2006). Yet we know less about how plant genotypic variation affects co- occurring pathogens, or “communities,” that include functionally and taxonomically diverse species, of large, small, or potentially synergistic effects on their plant hosts (Strauss et al. 2005; Morris et al. 2007). Because pathogens have the potential to define entire ecosystems (e.g., Chestnut blight fungus, Cryphonectria parasitica Russell 1987;

sudden oak death, Phytophthora ramorum Rizzo & Garbelotto 2003), it is important to elucidate the genetic and environmental factors shaping their natural communities. Plants possess two different types of genetic resistance that could affect pathogen communities: major-genes and quantitative genetic resistance. Major genes prevent infection by specific pathogens (Flor 1955), and thereby can potentially alter the composition (i.e., the presence or absence of particular pathogens) of the pathogen community of an individual plant. In contrast, quantitative genetic resistance, involves many genes acting together to reduce pathogen latent period, infection efficiency, or spore production (Geiger & Heun 1989). Because quantitative genetic resistance is neither complete nor pathogen-specific, it is likely to affect the abundance of pathogens within the community. Thus, individual plants are more likely to host divergent pathogen communities if major genes for resistance are important in structuring the composition of those communities. Alternatively, if quantitative, genetic resistance is more important, the relative abundance of each pathogen in the community may vary with it, but composition should remain unchanged. Host density could also influence pathogen communities. Reducing the relative abundance of susceptible hosts of a given pathogen can decrease disease transmission and severity. Also known as the dilution effect, this pattern has been observed in plant pathosystems with experimentally manipuated densities (Leonard 1969; Chin & Wolfe 1984; Mundt 1994; Newton et al. 1997; Zhu et al. 2000). If lower host densities are correlated with greater plant diversity, greater diversity in pathogen communities may also be expected (Strong et al. 1984; Siemann et al. 1998; Crutsinger et al. 2006; Johnson et al. 2006; Schweitzer et al. 2011). Populus is a foundation species of riparian ecosystems in Western North America (Whitham et al. 2003), and also the preeminent woody crop for biomass energy production (Rubin 2008; Sannigrahi & Ragauskas 2010). The genetic basis of resistance to many pathogens of Populus has been demonstrated; major resistance genes in particular are important for artificial hybrids (Newcombe 1996). However, little is known about Populus genetic resistance to entire pathogen communities. Here, we investigate how inter- and intraspecific genetic variation in Populus influences fungal leaf pathogen communities in a common garden where environmental variation is

minimized, and in natural forests throughout the Weber River drainage system (Utah, USA). This approach allowed us to evaluate whether genetic effects observed in common gardens can be scaled to whole watersheds. We hypothesized that plant species, and genotype within species, would be important for structuring pathogen communities. In addition, we hypothesized that the density of hosts in the wild would influence pathogen communities, reflecting that the dilution of a pathogen’s preferred hosts reduces its severity on that host.

3.3 Materials and methods

3.3.1 Study system Our study was conducted near Ogden, Utah, where Populus angustifolia, P. fremontii and their naturally occurring interspecific hybrid P. × hinckleyana are found in mixed stands along a 13 km stretch of the Weber River (hereafter hybrid zone) (Eckenwalder 1984). The overstory in these stands is dominated by Populus spp. representing approximately 70% of the individuals and 90% of the biomass (Adams et al. 2011). P. angustifolia occurs in pure stands at elevations above the hybrid zone (>1400m), and P. fremontii occurs in pure stands below the hybrid zone (<1300m). In 1991, a common garden was established within the hybrid zone. Replicated genotypes of P. angustifolia, P. fremontii and P. × hinckleyana were planted in the garden using cuttings taken from randomly selected trees growing along the Weber River in pure stands and in the hybrid zone. At the time of this study (September 2010) garden trees were sexually mature.

3.3.2 Pathogen community We define the fungal leaf pathogen community by those species causing visible symptoms of disease. A limited number of species fit this criterion, allowing us to examine plant species and genotype effects on individual pathogens in addition to our main objective of analyzing genetic effects on the entire pathogen community. Busby (Chapter 1) extensively sampled fungal pathogens on diseased leaves in the Weber River Populus hybrid system over two field seasons, and utilized both

morphological and DNA sequence data to identify the following commonly occurring taxa: Drepanopeziza populi, Phyllactinia populi and Mycosphaerella spp. (orders Helotiales, Erysiphales and Capnodiales, respectively). In this system, P. angustifolia is the most susceptible host for all three taxa (Chapter 1). Because a pattern of exapted resistance prevails in Populus, whereby non-hosts are more resistant to pathogens than preferred, native hosts (Newcombe 1998; 2005), we can infer that P. angustifolia is the preferred, native host for these taxa. In our pathogen community surveys for the present study, the three pathogen taxa were identifiable without magnification. Drepanopeziza populi was easily identified by its characteristic dendritic lesions and white acervuli (Fig 1A). In contrast, Mycosphaerella lesions were often angular, and the acervuli were black at the surface (Fig 1A). Phyllactinia populi mycelia and fruiting bodies were visible on the underside of leaves (Fig 1B). At the onset of the current study, three species of Mycosphaerella occurring in the Populus study systems had not yet been distinguished. M. angustifoliorum, M. musivoides and M. wasatchii remain indistinguishable in the field, and the latter two species have only recently been formally described (Chapter 1). We were unable to distinguish between species of Mycosphaerella in pathogen surveys for the present study, but we identified several specimens microscopically to determine their occurrence in pure P. angustifolia and P. fremontii stands, and in hybrid zone stands. Mycosphaerella and D. populi are necrotrophic (possibly hemibiotropic) pathogens: they kill host tissue and feed on the remains. These pathogens are known to cause reduced growth, premature defoliation, shoot and branch death, and stem cankers in Populus (Ostry 1987). Damage over multiple years can result in tree death (Ostry & McNabb 1986). P. populi is a biotrophic pathogen: it feeds on live host tissue. Phyllactinia populi may be less likely to affect Populus fitness because it occurs late in the growing season (Sinclair & Lyon 2005). There have been no formal studies of Populus genetic resistance to any of the specific pathogens evaluated in our study. However, major resistance genes are common for biotrophic pathogens like the powdery mildew fungus P. populi, whereas quantitative resistance is more common for necrotrophic pathogens like Mycosphaerella and D. populi (Oliver & Ipcho 2004). A previous study found evidence for quantitative

resistance to Mycosphaerella in Populus trichocarpa × P. deltoides (Newcombe & Bradshaw 1996), and we found evidence of quantitative, genetic resistance to D. populi in a gene expression study (P. Busby, unpub. data).

2.3.3 Pathogen communities in a common garden To test the hypothesis that Populus species and their hybrid, and genotypes within species, are characterized by distinct pathogen communities, we measured fungal pathogen severity on leaves collected from ten P. angustifolia genotypes, six P. × hinckleyana genotypes and four P. fremontii genotypes growing in a common garden. For each genotype we sampled 3-10 replicate clones. The uneven sample size of both clones and genotypes was an unavoidable constraint of the original garden design. Our pathogen community surveys consisted of scoring the severity of all pathogens present on multiple leaves of each tree sampled. Pathogen severity, as measured by damage, is strongly determined by plant genetic resistance in Populus (Newcombe & Bradshaw 1996). Since pathogens reproduce on damaged host tissue, severity is also a measure of pathogen abundance. Analysis of ecological communities typically utilizes presence/absence or abundance data on individual species within communities (McCune & Grace 2002). In our community analyses, we use pathogen severities for individual species as proxies for their relative abundance in the community. For each tree, we quantified the severity of each necrotrophic pathogen present by visually estimating leaf area damaged by that pathogen for 24 leaves standardized by leaf age (leaf plastochron index 3, 4, 5 and 6), collected from six haphazardly selected terminal shoots in the lower canopy. For all leaves, the severity of each pathogen was scored on a scale from 0 to 5 reflecting the percentage of leaf area damaged: 0 = no damage, 1 = 1-6%, 2 = 7-12%, 3 = 13-25%, 4 = 26-50% or 5 = >50%. Damage scores were then used to calculate a single weighted damage score (Dirzo & Domínguez 1995). Because the biotrophic pathogen P. populi did not cause tissue damage, this pathogen was scored as present or absent at the shoot level. In our surveys, we only recorded percent area damaged by Mycosphaerella or D. populi if diagnostic fruiting bodies were present; otherwise damage was recorded as caused by an unknown species. However, in most cases such lesions resembled

Mycosphaerella or D. populi with immature fruiting bodies. Therefore, our estimates of Mycosphaerella spp. and D. populi severity could be conservative, and damage caused by truly unknown species rare. Unknown pathogen damage could have been caused by Fusicladium romellianum (which was found infrequently but did not produce diagnostic characteristics during our summer survey), or other unidentified pathogens. Taken together, unknown damage accounted for approximately 25% of total recorded damage. We also used a complementary method to quantify pathogen communities in the common garden. Here, we used total percent leaf area damaged by all pathogens as a proxy for the pathogen community. We sampled total pathogen damage on 3-5 replicates of the same genotypes described above. For each tree, we: 1) photographed ten leaves with representative pathogen damage, and 2) estimated the percentage of leaves on each tree with any level of pathogen damage on a categorical scale (<25%, 26-50%, 51-75%, >76%). We imported photographs into GIMP image manipulation software, and used a digital tablet and pen to measure total necrotrophic pathogen damage as a percent of total leaf area. We then quantified tree-level severity as mean percent leaf pathogen damage multiplied by the categorical midpoint for the percentage of total leaves damaged.

2.3.4 Pathogen communities in the wild To determine if common garden results scale to whole watershed, we sampled pathogen communities on Populus angustifolia, P. fremontii and P. × hinckleyana in nineteen natural stands along the Weber River spanning lower elevation pure P. fremontii stands, through the hybrid zone, and into upper elevation pure P. angustifolia stands. In each natural stand, we haphazardly selected ten trees of each Populus taxon present. Pathogen communities on trees were sampled using the first method described above. In addition to evaluating genetic effects on pathogen communities in the wild, we evaluated how the density of the preferred host, P. angustifolia, affects pathogen communities. Stands within the hybrid zone are separated by natural or human made boundaries, and fall along a natural gradient in the density of Populus species and their hybrid: stands in closer proximity to the pure P. fremontii zone are more heavily dominated by P. fremontii, and vice versa for stands in closer proximity to the P. angustifolia zone, with P. × hinckleyana reaching its greatest abundance in stands located

near the middle of the hybrid zone (Wimp et al. 2004; Schweitzer et al. 2011). While each stand is characterized by different densities of both Populus species and their hybrid, otherwise stands are similar in size (1.8–2.6 ha2), overall Populus density (664.04 stems ha-1 ± 55.7 standard error), basal area (29.03 m2 ha-1 ± 4.07 standard error), climate, and soils (Schweitzer et al. 2011). We used the proportion of stand-level P. angustifolia alleles as a measure of the local density of the preferred host (Wimp et al. 2004). Because this is a hybrid system, 1- the proportion of P. angustifolia alleles is equal to the proportion of alleles for the non-preferred host, P. fremontii. Hybrids receive half of their alleles from each parental species. Data on environmental covariates (i.e., canopy openness (Adams et al. 2011) were also available for our analyses.

2.3.5 Statistical analyses All statistical analyses were conducted in R 2.8.1 (R Development Core Team 2008). We used permutational multivariate analysis of variance using distance matrices

(PERMANOVA, McArdle 2001) to: 1) estimate variance of pathogen communities in the common garden attributable to Populus species and their hybrid, and genotype (nested) within species, and 2) estimate variance in pathogen communities in the wild attributable to Populus species and their hybrid, and the proportion of P. angustifolia alleles. For the hybrid zone analysis, we included stand-level canopy openness as a covariate to account for environmental effects on pathogens that may be associated with differences in stand density. In particular, a closed canopy is more favorable to pathogen infection than an open canopy (Giesler & Yuen 1996). Stand-level canopy openness was calculated from the mean canopy openness determined at 12 randomly selected points within each stand (Adams et al. 2011). Our community matrices consisted of columns of pathogen severities, one for each species, excluding unknown pathogen species. We transformed severity data to ensure that all species contributed equally to community analyses. We arcsin transformed proportional (0-1) P. populi severity data (Zar 1996) and fourth-root transformed Mycosphaerella and D. populi percent damage data to eliminate high- scoring variables while preserving the weights (Clarke 1993). To test the significance of predictor variables, we used F-tests based on sequential sums of squares from

permutations of the raw data (McArdle 2001). To visualize community results, we used two-dimensional representations of pathogen communities where the x and y-coordinates are based on non-metric multidimensional scaling (NMDS) analysis. We also calculated principal component scores using the same community matrix to visualize the relationship between the proportion of P. angustifolia alleles and pathogen community structure. We used restricted maximum likelihood (REML, lme4 package) to estimate variance in individual pathogen severity attributable to species and genotype, and variance in the pathogen community attributable to host species and genotype for complementary image data (Falconer & Mackay 1996; Conner & Hartl 2004). Genotype was nested within host species in the common garden analysis, and both were random effects. The significance of genetic effects was tested using log-likelihood ratio tests (Conner & Hartl 2004). To evaluate and compare plant genotype effects among species, we calculated the total phenotypic variation in pathogen communities (and individual pathogens) explained by host genotype for each host species separately. This measure is called the broad sense 2 heritability (H = VarGenotype/[VarGenotype+VarError ]). To calculate the total phenotypic variation in pathogen communities we used PERMANOVA, and for individual species we used REML. Genotype was a random variable in all models; its significance for communities was tested using F-tests (McArdle 2001), and for individual species using log-likelihood ratio tests (Conner & Hartl 2004).

2.4 Results

2.4.1 Pathogen communities in the common garden Supporting our hypothesis that genetic differences between Populus species and their hybrid affect dependent pathogen communities, we found that Populus species and their hybrid explained 26-29% (p<0.001) of the variation in pathogen community structure (Table 1). In addition, we found support for the hypothesis that pathogen communities differ on different genotypes within species: host genotype explained 34- 38% (p<0.001) of the variance in pathogen community structure (Fig 2, Table 1). Broad-

sense heritability analyses within species revealed that the magnitude of genotypic effects on pathogens ranged from H2=0.33 for P. angustifolia, to 0.41 for P. × hinckleyana, to 0.75 for P. fremontii (Table 2). Overall, given that Drepanopeziza populi, Mycosphaerella spp. and Phyllactinia populi were the only pathogens found, and that they were present on almost all plant genotypes, our results show that pathogen communities varied mostly in the severities of pathogens rather than in composition (Fig 3).

2.4.2 Pathogen communities in the wild In the hybrid zone, we also found support for the hypothesis that genetic differences between Populus species and their hybrid affect dependent pathogen communities (Fig 4). However, Populus species and their hybrid explained less variation in pathogen community structure in the wild than in the common garden (R2=0.12, F=16.9, p<0.001) (Table 1). We also found support for the hypothesis that the density of the preferred host affects pathogen communities in the wild (R2=0.15, F=42.5, p<0.001) (Table 1). More specifically, the severity of pathogens on P. angustifolia declined with its density, resulting in a convergence in the structure of pathogen communities on Populus species and their hybrid in stands dominated by the non-preferred host, P. fremontii (Fig 5). Canopy openness did not influence pathogen community structure (F=1.3, p=0.26, Table 1), suggesting that observed differences in pathogen communities were not caused by environmental differences among stands. In agreement with our common garden results, pathogen communities in the wild varied mostly in the severity of pathogens rather than in composition (Fig 6). There were two exceptions. First, the biotrophic pathogen, Phyllactinia populi, was often absent from trees in the wild (Fig 6). And second, while all five pathogen species were observed on P. angustifolia and P. × hinckleyana, and four of the five occurred on P. fremontii in the hybrid zone, we observed only two pathogen species on P. fremontii in pure stands (Table 3).

2.5 Discussion

Both in a common garden and in the wild, we show that genetic variation in foundation tree species, and the density of a preferred host, can affect pathogen communities. While genetic effects were stronger in the common garden than in the wild, general agreement between results suggest that common garden results can scale to whole watersheds. These results support the overarching hypothesis that genotypic variation in foundation plant species (e.g., Populus spp, Solidago altissima, Oenothera biennis, Salix lasiolepis, Asclepias syriaca, Eucalyptus globulus) can strongly shape whole communities independent of taxonomic affinity. This work should have application for biodiversity conservation and restoration, as anthropogenic impact are increasingly homogenizing communities and populations (reductions in species richness/diversity, and genetic diversity, respectively).

2.5.1 Plant species effects on pathogens In common gardens, where environmental variation is controlled, different species of Populus and their hybrid support distinct arthropod communities (Wimp et al. 2005), and unidentified communities of soil microbes (Schweitzer et al. 2008), endophytes (Bailey et al. 2005) and mycorrhizal fungi (Gehring et al. 2006). For arthropods, the most well studied group, this effect is thought to be driven primary by herbivores which specialize on a preferred host by adapting to the specific, genetically controlled, structural and chemical defenses of that host (Bernays & Graham 1988). This was demonstrated in Populus, where plants that were more genetically similar were also more similar in secondary chemistry and arthropod communities (Bangert et al. 2005). We expected non-host resistance would drive differences in pathogen communities between Populus species and their hybrid (Wapshere 1974; Bernays & Graham 1988; Heath 2000; Gilbert & Webb 2007). However, all pathogen taxa were able to infect both Populus species and their hybrid. In agreement with our previous work (Chapter 1), we found that the severities of all pathogens – D. populi, Mycosphaerella and P. populi – were greatest on P. angustifolia, suggesting it is the preferred, native host. Within Mycosphaerella, P. angustifolia is the native host of M.

angustifoliorum and M. wasachii, while P. fremontii is the native host of M. musivoides (Chapter 1). D. populi, P. populi and M. angustifoliorum have shifted from native host P. angustifolia to novel host P. fremontii in the hybrid zone. Shifting from P. angustifolia to P. fremontii may rely on the genetic intermediate P. × hinckleyana and backcross hybrids (Martinsen et al. 2001). Possibly as a result of such step-wise shifting, we found that P. populi was most severe on native host P. angustifolia, intermediate on the hybrid P. × hinckleyana, and least severe on P. fremontii. These findings are consistent with the hybrid bridge hypothesis for host shifting in which hybrid intermediates facilitate host shifts among species (Floate & Whitham 1993). In pure P. fremontii stands we found less evidence of host shifting. P. populi and M. angustifoliorum were absent from P. fremontii in pure stands, but present on P. fremontii in hybrid zone stands. For these two pathogens, shifts to P. fremontii occurred only in the presence of P. angustifolia, P. × hinckleyana, and backcross hybrids. These results further support the hybrid bridge hypothesis.

2.5.2 Plant genotype effects on pathogens Differences in pathogen communities among genotypes were mostly in the severities of damage inflicted by consitituent pathogens rather than in composition. Because quantitative genetic resistance is thought to affect species abundances rather than composition, we infer that quantitative genetic resistance is more important than major genes for resistance for shaping these pathogen communities. Quantitative resistance is known to strongly affect necrotrophic pathogens, like D. populi and Mycosphaerella, and their severities were positively correlated in the co-occurrence on the same tree genotypes (R2= 0.41, p<0.0001). Alternatively, one of these pathogens could be directly or indirectly facilitating the other. The magnitude of genotypic effects on pathogens that we observed (H2 = 0.33- 0.75) is broadly similar to those reported for arthropods across foundation plant systems (e.g. H2=0.65 (Keith et al. 2010), H2=0.7 (Crutsinger et al. 2009), H2=0.41 (Johnson & Agrawal 2005). However, an explicit comparison between our results and previous work in the same Populus study system reveals that genotypic variation in P. angustifolia

explained twice as much variation in arthropod communities (65%, Keith et al. 2010) as pathogen communities (33%). The pattern of stronger plant genotype effects on arthropod than fungal pathogen communities is consistent with the results of a recent meta-analysis (Bailey et al. 2009). Fungal dependence on ambient moisture may be one reason why several studies in the Southwest, including ours, have not found stronger plant genetic effects on fungal communities (Gehring et al. 2006; Sthultz et al. 2009). In the case of fungal leaf pathogens, 12-72 hours of continual leaf wetness is required for infection (Agrios 2005), indicating that environmental factors will likely play a strong role in shaping pathogen communities.

2.6 Acknowledgements

We thank Gregory Gilbert, Megan Saunders and Joe Bailey for comments on an earlier draft of this manuscript. We also thank Alan Le and Denis Willet for their assistance with fieldwork. We are grateful for the Ogden Nature Center for hosting this research. This research was supported by the Department of Energy Global Change Education Program, the Heinz Environmental Fellowship, the Stanford Field Studies Program, and an NSF FIBR grant.

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2.8 Tables

Table 2.1 Model results (PERMANOVA) for pathogen community structure based on observational severity surveys in the common garden and in the wild, and image analysis (REML) of nectrophic pathogen damage in the common garden. Model results (REML) for individual pathogens are for the common garden only. For common garden analyses, genotype is nested within species for all models, and genotype and species are random effects. For REML models, the significance of species and genotype were tested using log-likelihood ratio tests, and for PERMANOVA using F-tests.

Hybrid zone analysis Host species P. angustifolia allele density Canopy openness R2 F p R2 F p R2 F p Pathogen community 0.12 16.9 <0.001 0.15 42.5 <0.001 0.005 1.3 0.26

Common garden analysis Host species Genotype R2 F p R2 F p Pathogen community 0.26 28 <0.001 0.34 4.2 <0.001 χ2 χ2 Pathogen community (images) 0.29 31.2 <0.0001 0.38 28.8 <0.0001 D. populi 0.15 13.3 0.0003 0.44 32.9 <0.0001 Mycosphaerella 0.41 41.6 <0.0001 0.23 22.9 <0.0001 P. populi 0.13 18.6 <0.0001 0.32 13.8 0.0002

Table 2.2 Proportion of variation in the structure of pathogen communities, and in individual pathogen attack severities, explained by genotype for different Populus species and their hybrid (broad-sense heritability, H2). For individual pathogens and community image data we used REML models, for the community we used PERMANOVA.

D. populi Mycosphaerella spp. P. populi Host species H2 χ2 p H2 χ2 p H2 χ2 p P. fremontii 0.33 1.8 0.18 0 <0.001 0.9 0.99 51.8 <0.001 P. × hinckleyana 0 1 1 0.58 10.7 0.0012 0.39 5.6 0.02 P. angustifolia 0.55 20.9 <0.0001 0.41 12.8 0.003 0 0 1

Community Community (images) Host species H2 F p H2 χ2 p P. fremontii 0.75 12.8 0.002 0.48 3.6 0.06 P. × hinckleyana 0.41 3.3 0.008 0 0 1 P. angustifolia 0.33 2.8 <0.001 0.54 15.4 <0.001

Table 2.3 Foliar fungal pathogens found on Populus species and their hybrid in natural stands along the Weber River, Utah. Ten trees per host were sampled in each of five stands in the pure P. fremontii zone, six stands in the hybrid zone, and eight stands in the pure P. angustifolia zone (N=310 trees).

P. fremontii P. pure Hybrid angustifolia zone zone pure zone

P. P. P. × P. P. Order Genus Species fremontii fremontii hinckleyana angustifolia angustifolia

Capnodiales Mycosphaerella angustifoliorum + + + + Capnodiales Mycosphaerella wasatchii + + + + Capnodiales Mycosphaerella musivoides + + + + + Helotiales Drepanopeziza populi + + + + + Erysiphales Phyllactinia populi + + + +

2.9 Figures

Fig 2.1 Fungal leaf pathogens of the hybrid Populus study system. A) Mycosphaerella lesion with black acervuli (right) and Drepanopeziza populi dendritic lesions with white acervuli (left), and B) magnified Phyllactinia populi mycelia and mature fruiting bodies.

Fig 2.2 Two-dimensional representation of pathogen communities found on ten P. angustifolia genotypes, four P. fremontii genotypes and six P. × hinckleyana genotypes in the common garden. Coordinates are based on global, non-metric multidimensional scaling (NMDS) analysis. Error lines are standard error of the mean.

Fig 2.3 Pathogen severity in the common garden for Drepanopeziza populi (A, D) Mycosphaerella spp. (B, E) and Phyllactinia populi (C, F) for P. angustifolia, P. fremontii and P. × hinckleyana. Left panels show differences between species (different letters indicate significant differences, p<0.01); right panels show differences among genotypes within species (N=4 for P. fremontii, N=6 for P. × hinckleyana and N=10 for P. angustifolia).

Fig 2.4 Two-dimensional representation of foliar pathogen communities found on Populus angustifolia, P. fremontii and P. × hinckleyana in the hybrid zone natural stands along the Weber River (N=70 per Populus host). Coordinates are based on global, non- metric multidimensional scaling (NMDS) analysis (stress = 0.11). Error lines are standard error of the mean.

Fig 2.5 Relationship between the density of stand-level P. angustifolia alleles and pathogen community structure (measured by PCA) for Populus angustifolia, P. fremontii and P. × hinckleyana in six hybrid zone stands.

Fig 2.6 Pathogenic severity for Drepanopeziza populi, Mycosphaerella, and Phyllactinia populi in natural stands along the Weber River. Severity for D. populi and Mycosphaerella is percent leaf area infected; severity for P. populi is the proportion of shoots infected. Grey bars mark the boundaries of the hybrid zone. Error bars are stand error of the mean.

Chapter 3

Effects of plant genotype on associated pathogen community attenuate as environmental heterogeneity increases

Posy E. Busby,1* George Newcombe2, Rodolfo Dirzo1 and Thomas G. Whitham3

3.1 Abstract

Genotypic differences in plants can result in different outcomes for their interactions with associated species and entire communities. Here, we test whether such genetic effects depend on the degree of environmental heterogeneity. In a reciprocal transplant experiment, using common gardens in three contrasting environments along an elevational gradient, we determined the importance of Populus angustifolia genotype for structuring fungal leaf pathogen communities composed of taxa from three Orders. We found that plant genotype played a significant role in structuring pathogen communities locally: for two consecutive years, genotype explained 23-40% of the variation in pathogen community structure within all three gardens. But among gardens, environment explained more than twice as much variation (29%) in pathogen community structure as genotype (12%) and genotype by environment interaction (GxE, 10%). To evaluate potential mechanism(s) underlying the GxE interaction, we conducted a reciprocal inoculation greenhouse experiment. Seedlings from six P. angustifolia populations located along the same elevational gradient were inoculated with a single pathogen species (Drepanopeziza populi) collected from three of those populations. We found evidence for a selection pressure gradient influencing genetic resistance: seedlings originating from low-elevation environments where disease risk is high were more resistant than seedlings originating from high-elevation environments where disease risk is low. Overall, our findings indicate that plant genotypic influences on associated pathogen communities can be important locally, but attenuate with increasing environmental heterogeneity, and that the environmental setting strongly affects both the plant’s associated pathogen community and the level of genetic resistance to pathogens.

3.2 Introduction

Many studies have shown that genetic differences in plants resulting from hybridization, polyploidization, and intraspecific genetic differences within populations, can result in major differences for associated species (Antonovics 1992; Thompson et al. 1997; Agrawal 2003; Whitham et al. 2003; Sinclair & Lyon 2005; Johnson et al. 2006; Hughes et al. 2008; Adams et al. 2011). Genotypic variation within foundation species, or those that create locally stable conditions for many associated species (Dayton 1972), can be particularly influential for dependent species (Whitham et al. 2003). For example, Populus spp., a set of foundation tree species typical of riparian forests in western North America, has strong genotypic effects on arthropods, birds, and soil microbes within common gardens where environmental heterogeneity is minimized (Whitham et al. 2006). However, for many plant systems, it is not known how genotypic effects on associated communities are influenced by environmental variation in natural landscapes (Johnson & Agrawal 2005; Tack et al. 2010) One hypothesis is that environmental variation overwhelms genetic effects on dependent communities at larger spatial scales that are inherently more heterogeneous (Bangert et al. 2008). A second hypothesis, the geographic mosaic theory of coevolution (Thompson 2005), argues that genetic influences on species interactions vary along environmental gradients (Thompson 1997). In other words, species interactions associated with genotypes of foundation species depend on the environment in which they occur (genotype by environment, or GxE interaction). As environmental conditions change, so do species abundances and selection pressures, resulting in different outcomes for species interactions. Evaluating these hypotheses in natural plant systems can help clarify the sensitivity of plant genotypic effects to environmental heterogeneity. Interactions between plants and pathogens are known to be heavily influenced by genotypic variation in plant resistance (Jones & Dangl 2006). Foliar pathogens reduce leaf area and associated photosynthetic activity, and thereby negatively affect plant growth and reproduction. In response, plant populations may undergo selection for resistant genotypes (Gilbert 2002). Two distinct types of induced genetic resistance are found in plants: quantitative resistance, which involves many genes acting together to

reduce pathogen latent period, infection efficiency, or spore production (Geiger & Heun 1989) and gene-for-gene (GFG) resistance, whereby pairs of matching genes control both plant resistance and pathogen virulence (Flor 1955). Quantitative resistance is thought to have similar effects on a broad range of pathogens, particularly those that derive energy from dead plant cells (necrotrophs), while GFG resistance is more specific to individual pathogens that derive energy from living plant cells (biotrophs) (Oliver & Ipcho 2004). There has been extensive study of spatial variation in the relationship between plants and particular biotrophic pathogens (e.g., Linum marginale–Melampsora lini, Silene alba– Ustilago violacea, Filipendula ulmaria–Triphragmium ulmariae, and Valeriana salina– Uromyces valerianae) (Burdon & Thrall 1999). However, our knowledge of spatial variation in plant genetic effects on entire pathogen communities is not well developed. Collective evidence from hundreds of studies of host-pathogen resistance indicates that plant genotype can outweigh environmental effects on individual pathogens (Parker 1985; Burdon & Jarosz 1991; Jarosz & Burdon 1991). However, a bias toward studying single, damaging, and often biotrophic pathogens may have led to an inflated perception of plant genetic effects on pathogens in natural systems. Furthermore, studies are typically carried out in systems where negative effects on host fitness are operative, and where study environments are conducive to infection, such as plantations. Both spatial variation in the strength of reciprocal selection between plants and pathogens (Prakash & Heather 1986; Geiger & Heun 1989; Jarosz & Burdon 1991; Kilian et al. 1997; Laine 2005), and the occurrence of environments conducive to infection, should determine the impact of plant genetics on pathogen communities (Burdon & Thrall 1999). A recent study found that Populus angustifolia genotype explained 33-54% of the variation in fungal leaf pathogen communities dominated by necrotrophic pathogens within a single common garden environment, in a single year (Chapter 2). Genotypic variation in resistance also modulates the indirect, negative effects of pathogens on foliar arthropod communities (Chapter 4). Because pathogens in this system, and others (Russell 1987; Rizzo & Garbelotto 2003) can be keystone species defining much larger communities and ecosystems, we sought to determine the extent to which plant genotypic effects on pathogen communities depend on environmental heterogeneity.

For two consecutive years (2009-10), we sampled fungal leaf pathogen communities on P. angustifolia genotypes reciprocally planted in three common gardens located along an elevation and distance gradient (55 km) on the Weber River in Utah, USA. We first test the hypothesis that plant genotypic effects on pathogen community structure (composition and species severities) are operative locally, within contrasting common garden environments. Inter-annual variation can strongly influence pathogens (Burdon & Thrall 1999), so we compared our results for two different field seasons to evaluate their consistency. Next, we combine data from all common garden environments and evaluate the importance of plant genotype (G), environment (E), and their interaction (GxE) for structuring pathogen communities. Here, we test the hypothesis that genetic influences on community structure attenuate at larger spatial scales that are inherently more heterogeneous. Finally, to evaluate potential mechanism(s) underlying GxE interactions, we conducted a reciprocal inoculation greenhouse experiment using seedlings from six P. angustifolia populations located along the same elevational gradient, and inoculum from a single pathogen species (Drepanopeziza populi) collected in three of those populations.

3.3 Materials and methods

3.3.1 Study system Our study was conducted in the Wasatch Mountains in north-central Utah, where the narrowleaf cottonwood, Populus angustifolia, occurs along upper reaches of the Weber River. This is a relatively arid region where opportunities for pathogens to infect hosts, and affect host fitness, may be limited. Three common gardens, hereafter A, B and C, were established in 1991 along the river at 1300 m, 1392 m, and 1587 m. The gardens were reciprocally planted with replicated P. angustifolia genotypes using cuttings collected from trees growing in natural stands along the same stretch of the Weber where gardens are located. By the time of our study (2009-10) garden trees were sexually mature. To characterize environmental differences among gardens, we simultaneously measured temperature and relative humidity every 10 minutes for two weeks in August

and September 2010 using 5-7 HOBO® sensors in each garden. We calculated mean daily values over this time period for each parameter. Garden B, located in an exposed environment at the entrance to Weber Canyon, was the hottest and driest garden (Fig 1). Gardens A and C had similar daytime temperatures and relative humidity, but the highest elevation garden, C, was significantly cooler at night (Fig 1).

3.3.2 Pathogen community We define the fungal leaf pathogen community by those species causing visible symptoms of disease. Busby (Chapter 1) used morphological and DNA sequence data to characterize this community. Common pathogens in the study area are all Ascomycota: Drepanopeziza populi, Phyllactinia populi and Mycosphaerella spp. (orders Helotiales, Erysiphales and Capnodiales, respectively). These taxa are identifiable without magnification. D. populi was easily identified by its characteristic dendritic lesions and white acervuli (Fig 2A); lesions of Mycosphaerella were contrastingly angular and characterized by black acervuli (Fig 2A). P. populi mycelia and fruiting bodies are also readily visible on the underside of leaves (Fig 2B). However, at the time of our sampling for the current study, we were unaware of several species of Mycosphaerella that were indistinguishable in the field and previously undescribed (M. angustifoliorum, M. wasatchii, and M. musivoides). Therefore, we were not able to distinguish between species of Mycosphaerella for the present study. Mycosphaerella and D. populi are necrotrophic (or hemibiotrophic) pathogens that kill host tissue and feed on the remains. They are known to cause reduced growth, premature defoliation, shoot and branch death, stem cankers, and eventual death in Populus (Ostry & McNabb 1986; Ostry 1987). In contrast, P. populi is a biotrophic pathogen that feeds on live plant tissue. This powdery mildew fungus may be less likely to affect host fitness because it occurs late in the growing season (Sinclair & Lyon 2005). We assessed the potential for pathogens to affect Populus performance by examining the relationship between tree-level pathogen severities and premature leaf abscission, shoot length, and shoot diameter for the current year. Premature leaf abscission was quantified as the number of leaf scars divided by the total number of leaves present plus scars, for

all actively growing terminal shoots collected. These data were collected at the same time as pathogen community surveys (see below). The pathogens of P. angustifolia are ideal for studying community-level responses because their numbers are tractable, which allowed us to additionally examine genotype effects on individual pathogens. Analysis of ecological communities typically utilizes presence/absence or abundance data on individual species within communities (McCune & Grace 2002). We use pathogen severities for individual species as proxies for their relative abundance in the community. Pathogenic severity in Populus is strongly influenced by plant genetic resistance (Newcombe & Bradshaw 1996), but should also depend on the suitability of the environment for pathogen development (Gilbert 2002). Since pathogens reproduce on damaged host tissue, severity of damage should be correlated with pathogen abundance.

3.3.3 Reciprocal transplant experiment Our pathogen community surveys consisted of scoring the severity of damage for all pathogens present on multiple leaves of each tree sampled. In late summer (September), when foliar pathogens of Populus are at their peak severities, we measured fungal pathogen damage on leaves collected from five P. angustifolia genotypes that were reciprocally planted across gardens. In each garden, for 3-10 replicates of each P. angustifoila genotype (utilizing all the clones available), we estimated tree-level severity for each pathogen by visually quantifying leaf area damaged for 18-24 leaves standardized by age (leaf plastochron index 3, 4, 5 and 6), collected from 6 haphazardly selected terminal shoots in the lower canopy. For all leaves, damage for each necrotrophic pathogen was scored on a scale from 0 to 5 reflecting the percentage of leaf area damaged: 0 = no damage, 1 = 1-6%, 2 = 7-12%, 3 = 13-25%, 4 = 26-50% or 5 = >50%. Damage scores were then used to calculate a single weighted damage score (Dirzo & Domínguez 1995). We also scored damage for unknown pathogens, which were those without diagnostic features. Unknown damage accounted for an average of 14% of all observed pathogen damage. In most cases, we speculate unknown pathogens were Mycosphaerella or D. populi with immature fruiting bodies. Alternatively, unknown pathogen damage could have been caused by Fusicladium romellianum, which

occurred infrequently in the study area but did not produce diagnostic characteristics during our surveys, or other unidentified pathogens. Due to the absence of necrotrophic tissue caused by the biotrophic pathogen P. populi, this pathogen was scored as present or absent at the shoot level in both years. To examine spatial variation in plant genotypic effects on pathogens within contrasting common garden environments, we calculated the proportion of total phenotypic variation in pathogen communities, and individual pathogens, explained by P. angustifolia genotype. This metric is called the broad sense heritability 2 (H =VarGenotype/[VarGenotype+VarError]), and is calculated for populations of trees within specific environments (Shuster et al. 2006). For each garden, we calculated H2 for 2009 and 2010 using two different datasets; first, including only the five P. angustifolia genotypes that are found in all three gardens, and second, including additional genotypes that were found in only one or two gardens, therefore not utilized in across-garden analysis. In total, we sampled ten P. angustifolia genotypes in the garden A, ten genotypes in garden B, and seven genotypes in garden C. Next, we combined the data from all gardens to test the relative importance of plant genotype (G), environment (E) and genotype by environment interaction (GxE) for pathogen community composition, and for individual pathogen severities. We also included study year (Y) and genotype by year interaction (GxY) in analytical models to evaluate the importance of seasonal (i.e., inter-annual) variability for pathogen community composition. Statistical analyses were conducted in R 2.8.1 using the lme4 and vegan packages (R Development Core Team 2008). We used permutational multivariate analysis of variance (PERMANOVA) using distance matrices to estimate the variance in pathogen community composition explained by plant genotype within gardens, and the variance in composition among gardens (cf. McArdle 2001) attributable to G, E, GxE, Y and GxY. Our community matrices consisted of columns of pathogen severities, one for each species, excluding unknown species. We transformed pathogen severity data, to ensure that each species contributed equally to community analysis, as follows: we fourth-root transformed Mycosphaerella and D. populi data to eliminate high-scoring variables while preserving the weights (Clarke 1993), and arcsin-transformed proportional (0-1) P. populi

data (Zar 1996). To test the significance of each factor we used F-tests based on sequential sums of squares from permutations of the raw data (McArdle 2001). We used restricted maximum likelihood (REML) models to estimate the variance in individual pathogenic severities explained by plant genotype, where genotype was a random factor, and its significance was tested using log-likelihood ratio tests (Falconer & Mackay 1996; Conner & Hartl 2004). We also used REML to estimate variance in individual pathogen severity attributable to G, E, GxE, Y and GxY. In the full model Y was a fixed effect, and G, E, GxE and GxY were random effects. The significance of Y was tested using an F-test; the significance of G, E, GxY and GxE were tested using log- likelihood ratio tests (Falconer & Mackay 1996; Conner & Hartl 2004).

3.3.4 Reciprocal inoculation experiment To evaluate the potential mechanism(s) underlying GxE interaction effects, we conducted a reciprocal inoculation experiment. For this experiment, we selected a single pathogen that as found to be strongly influenced by GxE in the reciprocal transplant experiment, Drepanopeziza populi. We reciprocally inoculated P. angustifolia seedlings collected from six natural stand populations using pathogen inoculum collected from three of those populations. Five populations were located along the same Weber River elevation gradient; one population was located in a low-elevation stand on the Ogden River, a tributary of the Weber. In each stand natural stand, we assessed the level of disease risk by sampling pathogen severity on ten haphazardly selected P. angustifolia mature trees using the severity index described above. In July 2010, P. angustifolia seed was collected from a single female tree in each population. Seedlings (half-sibs) were raised in a greenhouse at Stanford University, California. In total, we generated an average of 74 half-sibs per population (range = 30- 140). At the same time (July), we collected leaves infected with the pathogen D. populi from at least five P. angustifolia individuals in three of the six populations: a low (1581 m) and high elevation (2058 m) population along the Weber River, and a low elevation Ogden River population (1586 m). Leaves were moist-incubated for one week to stimulate asexual spore release. Spores were suspended in deionized water and stored frozen. The concentration of inoculum solutions was approximately 70,000 D. populi

conidia per ml (upper-elevation Weber River = 72,000; lower-elevation Weber River = 69,000; Ogden River = 65,000). Three-month-old seedlings were inoculated with the pathogen by spraying spore suspensions on leaves, and maintaining moisture on the leaf surface for 12 hours. We inoculated an average of 29 seedlings per host population/pathogen combination (range = 5-56). We collected data on pathogen severity two weeks after inoculation. For each individual, we photographed leaves with plastochron index 3, 4 and 5, and used Image J to quantify the percentage of leaf area infected. Color and brightness filters were used to transform photographs into binary, black and white images with pathogen damage in white and healthy leaf material in black. We calculated mean leaf area damaged (%) for each individual, and divided this number by the mean for each inoculum solution. This relative value allowed us to compare average population-level damage across inoculum solutions. Finally, for each inoculum solution, we used linear regression to determine the proportion of variation in relative pathogen damage explained by the elevational gradient.

3.4 Results

3.4.1 Reciprocal transplant experiment In agreement with the hypothesis that plant genotypic effects on pathogen community structure are operative locally, we found that plant genotype explained 23- 40% of the variation in pathogen community composition within different gardens and years (Table 1). However, genotypic effects on individual pathogens were variable among species, gardens and years (Table 1). H2 for P. populi was never significant because all genotypes were either nearly uniformly infected or not, depending on the environment (Table 1, Fig 3). H2 for D. populi was greater in 2010 than 2009 in gardens B and C, and generally declined with the elevation of the common garden (Table 1, Fig 3). H2 for Mycosphaerella was significant and relatively stable across gardens in both years (Table 1, Fig 3). Overall, whether H2 was greater for D. populi or Mycosphaerella varied both spatially and temporally. In agreement with the hypothesis that plant genotypic influences on pathogen community structure attenuate as spatial scale and the effects of environmental

heterogeneity increase, among gardens we found the effect of plant genotype on pathogen composition was reduced by approximately 66%. E explained more than twice as much variation (29%, p<0.001) in pathogen community composition as G (12%, p<0.001), GxE interaction (10%, p<0.001) and Y (7%, p<0.001) (GxY not significant, Table 2). For individual pathogens, P. populi was most strongly influenced by E; D. populi and Mycosphaerella were most strongly influenced by GxE (Table 2). Study year was significant for D. populi and Mycosphaerella, but not for P. populi (Table 2). GxY was not significant for the community or any individual pathogen (Table 2). Within gardens, we found evidence that D. populi and Mycosphaerella negatively affect P. angustifolia performance, but no evidence that P. populi affects performance (Table 3). In both years, we found a positive correlation between D. populi and Mycosphaerella severity and premature defoliation in garden A, where pathogen severities were highest (Table 3). In garden A, we also found evidence for a negative effect of Mycosphaerella on stem length in 2009, and a negative effect of Mycosphaerella on stem diameter in 2010 (Table 3). There was less evidence of pathogens affecting plant performance in gardens B and C. In 2009 only, there was a positive relationship between Mycosphaerella severity and premature defoliation in garden C (Table 3). We found no relationship between D. populi or Mycosphaerella severity and premature defoliation in the garden B (Table 3), and no relationship between pathogen damage and the diameter or length of shoots in gardens B or C (Table 3).

3.4.2 Reciprocal inoculation experiment Results of the reciprocal inoculation experiment shed light on the mechanisms underlying observed GxE interaction effects on pathogens. In the inoculation experiment, damage caused by the pathogen D. populi increased with the elevation at which populations originated (F=72.9, R2=0.14, p<0.001) (upper panel, Fig 4). In natural stands, pathogen severity, or “disease risk,” decreased with elevation (lower panel, Fig 4). These responses to elevation were correlated: plants from low-elevation environments characterized by high pathogen damage were more resistant across all inoculum solutions, whereas plants from high-elevation environments characterized by low pathogen damage more susceptible across all inoculum solutions.

3.5 Discussion

Many studies have shown that genotypic variation in foundation plant species can strongly influence dependent communities within controlled environments (Antonovics 1992; Neuhauser et al. 2003; Agrawal 2004; Keith et al. 2010), and we are beginning to better understand how these effects vary across heterogeneous landscapes (Thompson 1997; Burdon & Thrall 1999; Craig et al. 2007; Soubeyrand et al. 2009; Smith et al. 2011). For example, separate origins of Heuchera polyploidy across the mountains of Idaho differentially shape herbivore and pollinator communities (Thompson 1997). And genotypic variation in P. angustifolia resistance to aphids influences predation by birds and their top-down effects differently in different environments (Smith et al. 2011). Our study evaluated how environmental heterogeneity affects the relationship between P. angustifolia genotype and its pathogen community. While we consistently found plant genotype effects on pathogen communities at local scales, the role of plant genotype in shaping communities at the landscape-scale was reduced by approximately 66%. Overall, our results indicate that the influence of plant genotype on pathogen communities will likely be stronger at local scales than across heterogeneous landscapes, and that plant genetic resistance to pathogens is correlated to the prevalent level of disease risk in the environment where it adapted resistance.

3.5.1 Genotypic effects at the local scale While we consistently found genotypic effects on pathogen communities within common garden environments, the structure of those communities varied. The identity of the most severe pathogen, in terms of observed severity and potential fitness consequences, was not always the same in different common gardens, or in different natural stands (Supp. Fig 1). This is relevant, because severe pathogens were strongly influenced by plant genotype, and therefore were major drivers of plant genotypic effects on the community overall. For example, in garden C, genotypic effects on Mycosphaerella (H2=0.35-0.42) greatly outweighed those for D. populi and P. populi (H2=0-0.19). But in garden A, genotypic effects on D. populi (H2=0.56-0.64) outweighed

those for Mycosphaerella and P. populi. (H2=0-0.4). These patterns suggest a geographic mosaic of interactions between plants and pathogens that is defined by pathogen-specific “hotpots” (D. populi in garden A, Mycosphaerella in garden C) and “coldspots” (P. populi in garden B, D. populi in garden C) for pathogen development. Asynchronous patterns of pathogen colonization and extinction may also influence this mosaic (Burdon & Thrall 1999).

3.5.2 Genotypic effects at the landscape scale At the landscape scale, we found the effect of genotypic variation on pathogen communities was reduced by approximately 66%. The magnitude of the decline was the same, whether we used five P. angustifolia genotypes for our analysis or included the additional genotypes found in only one or two gardens (data not shown). This result supports the hypothesis that plant genetic influences on community organization attenuate as spatial scale and environmental heterogeneity increase (Bangert et al. 2008). Our results are consistent with previous studies showing diminished genotypic effects on arthropods (Stiling & Rossi 1995; Johnson & Agrawal 2005; Bangert et al. 2006; Tack et al. 2010), and unidentified communities of mycorrhizal fungi (Gehring et al. 2006) and macrofungal decomposers at intermediate spatial scales (Barbour et al. 2009). The influence of plant genotype on pathogen communities among gardens was likely, in part, diminished because larger spatial scales include less suitable habitat for pathogen development and the expression of genetic resistance (i.e. H2 was lowest in high elevation garden C). Environmental factors affecting pathogen development could include abiotic and/or biotic environmental differences among gardens. Climatic differences among gardens could affect pathogen performance. For example, mildews are particularly sensitive to environmental conditions (Burdon & Chilvers 1982) and P. populi was almost entirely absent from the dry, and windy garden B in both years. Likewise, a negative relationship between D. populi and garden elevation suggests cold temperatures may limit D. populi. Finally, biotic “environmental” effects could include the presence or absence of plant endophytes that affect pathogen resistance (Arnold et al. 2003; Ganley et al. 2008); such endophytes are known in P. angustifolia (P. Busby unpub. data) and warrant further examination.

While E most strongly shaped pathogen communities at intermediate scales, G also played a significant role. Plant genotypic effects on pathogen communities dominated by necrotophic pathogens, like ours, may result from pathogens responding similarly to quantitative genetic resistance (Chapter 2). Supporting this hypothesis, D. populi and Mycosphaerella spp. were positively correlated in the co-occurrence on the same tree genotypes in garden A in 2009 and 2010, and in garden C in 2010 (data not shown). The influence of plant genotype on pathogen communities among gardens was also reduced as a result of plant genotype affecting pathogens differently in different environments. A GxE interaction has long been interpreted as an indication of local adaptation (or maladaptation) (Clausen et al. 1948). For example, tree genotypes may be most resistant in environments that most closely resemble the environment where they adapted resistance. Alternatively, pathogen populations may be locally adapted or maladapted to tree populations. In this case, pathogen populations in each garden would perform best or worst on plant genotypes that are most genetically similar to plant genotypes in natural stands surrounding the particular garden. This will differ garden-by- garden if plant genotypes are collected from different populations and reciprocally planted, as they were in our study (P. angustifolia genotypes were collected from two distinct stands, three genotypes from a stand closer to garden A, two genotypes from a stand closer to garden B). Our reciprocal inoculation results did not support local pathogen adaptation to tree populations, or vice versa. Instead, we found evidence that genetic resistance within tree populations is strongly influenced by environmental heterogeneity (the elevational gradient). Trees originating from populations characterized by high disease risk were more resistant, while trees originating from populations characterized by low disease risk were more susceptible. These results strongly suggest that plant genetic resistance may be adapted to the level of disease pressure supported by the particular environment.

3.6 Conclusions

Our study supports both the scale-dependent hypothesis (Bangert et al. 2008) and the geographic mosaic theory of coevolution (Thompson 2005). We found plant genotypic effects on pathogen communities attenuate as spatial scale and environmental heterogeneity increase along an elevational gradient. We also found evidence of a selection pressure gradient affecting genetic resistance to pathogens. These results illustrate how an elevational gradient can directly affect pathogen communities, and influence the level of plant genetic resistance to pathogens. We expect that our results will have conservation implications if, as predicted (Harvell et al. 2002), rapid climate change shifts environments and associated disease pressure outside of the range to which plants have adapted. If such changes favor particular plant genotypes, the ecology of associated pathogen communities may also be affected.

3.7 Acknowledgements

We thank John Thompson, Tadashi Fukami and Rachel Adams for comments on an earlier draft of this manuscript. We also thank Alan Le and Denis Willet for their assistance with field work. We are grateful for the Ogden Nature Center for hosting this research. This research was supported by the Department of Energy Global Change Environmental Program, the Heinz Environmental Fellowship, the Stanford Field Studies Program, and an NSF FIBR grant.

3.8 References

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3.9 Tables

Table 3.1 The proportion of variation in pathogen severities and pathogen community composition explained by plant genotype (H2), and results of tests for the statistical significance of plant genotype.

Garden Garden Garden A B C 2010 H2 χ2 p H2 χ2 p H2 χ2 p Mycosphaerella 0.41 13 0.005 0.26 11.6 0.009 0.42 25.2 <0.0001 D. populi 0.56 21.4 <0.0001 0.31 15.8 0.001 0.19 8.1 0.04 P. populi 0 1.4 0.71 NA 0 2.4 0.49 H2 F p H2 F p H2 F p Community 0.33 2.8 <0.001 0.37 2.9 <0.001 0.29 2.3 <0.001

2009 H2 χ2 p H2 χ2 p H2 χ2 p Mycosphaerella 0.4 21.6 <0.0001 0.38 21.8 <0.0001 0.35 20.2 0.0002

D. populi 0.64 58 <0.0001 0.26 9.6 0.02 0 2.6 0.46 P. populi 0 2.8 0.42 0 2.6 0.46 0 2.4 0.49 H2 F p H2 F p H2 F p Community 0.4 3.8 <0.001 0.35 2.8 <0.001 0.23 2 0.039

Table 3.2 REML and PERMANOVA results revealing the proportion of variance in pathogen severities and pathogen community composition explained by genotype (G), environment (E), genotype by environment interaction (GxE), year (Y) and genotype by year interactions (GxY). Dependent Independent Variance χ2* p variable variable Mycosphaerella Genotype 0.048 19.9 <0.001 Environment 0.15 28.9 <0.001 GxE 0.3 29.2 <0.001 Year 27.9 <0.001 GxY 0 0.99 D. populi Genotype 0.019 3.9 0.048 Environment 0.19 25.4 <0.001 GxE 0.22 9.02 0.003 Year 4.4 0.036

GxY 0 0.98 P. populi Genotype 0 0.99 Environment 0.75 171 <0.001 GxE 0 0.99 Year 2.1 0.15 GxY 0 1 R2 F p Community Genotype 0.12 10.5 <0.001 Environment 0.29 52.9 <0.001 GxE 0.096 4.4 <0.001 Year 0.077 27.4 <0.001 GxY 0.002 0.23 1 * F for Year

Table 3.3. Linear regression model results showing the variance in premature leaf abscission, stem diameter, and stem length for the current year explained by pathogen severity. Phyllactinia populi severity did not differ between genotypes so was not used in this analysis. 2009 D. populi Mycosphaerella N trees Garden A R2 p R2 p 137 Premature leaf abscission 0.29 <0.001 0.33 <0.001 Stem diameter ns ns Stem length ns 0.05 0.01 Garden B R2 p R2 p 57 Premature leaf abscission ns ns Stem diameter ns ns Stem length ns ns Garden C R2 p R2 p 47

Premature leaf abscission ns 0.39 <0.001 Stem diameter ns ns Stem length ns ns 2010 Garden A R2 p R2 p 129 Premature leaf abscission 0.44 <0.001 0.07 0.003 Stem diameter ns 0.07 0.005 Stem length ns ns Garden B R2 p R2 p 59 Premature leaf abscission ns 0.07 0.03 Stem diameter ns ns Stem length ns ns Garden C R2 p R2 p 47 Premature leaf abscission ns ns Stem diameter ns ns Stem length ns ns

3.10 Figures

Fig 3.1 Temperature (C°) and relative humidity (%) within common gardens A, B and C. Bars are standard errors of means for values collected daily for two consecutive weeks in August and September 2010 using HOBO® dataloggers.

Fig 3.2 Common fungal leaf pathogens of Populus angustifolia in the Utah study area: A) D. populi dencritic lesion with white acervuli (left) and angular Mycosphaerella lesion with black acervuli (right), and B) P. populi mycelia and mature fruiting bodies.

Fig 3.3 Plots showing mean pathogen severity for D. populi, Mycosphaerella spp. and P. populi in three common garden environments. Different colors represent different host genotypes. Error lines are standard errors of means.

Fig 3.4 Upper panel shows D. populi severity on reciprocally inoculated P. angustifolia populations. Elevation and distance (in km) between host populations located along the Weber River is depicted along the x-axis. Data points show mean pathogen damage (with standard error) for tree populations inoculated with each of the three inoculum solutions (i.e. low-elevation Weber River, high-elevation Weber River, and low-elevation Ogden River). Values greater than one indicate above-average population-level damage for the particular inoclum solution; values less than one indicate below-average damage. Lower panel shows observational data on disease risk in the same natural stands, collected in September 2010.

3.11 Supplementary Figures

Fig 3.S1 Pathogen severity for D. populi, Mycosphaerella and P. populi in sixteen natural stands (black circles) in 2009 and 2010. Grey squares indicate severity of pathogens in the gardens A, B and C. Bars are standard errors of means.

Chapter 4

Plant genetic resistance modulates the indirect effects of pathogens on arthropod communities and herbivory in foundation trees

Posy E. Busby,1* Louis J. Lamit, 2 Arthur Keith, 2 George Newcombe, 3 Thomas G. Whitham2 and Rodolfo Dirzo1

1Stanford University, Department of Biology, 371 Serra Mall, Stanford CA 94305 USA 2Department of Biological Sciences, Northern Arizona University, Flagstaff AZ 86011 USA 3College of Natural Resources, University of Idaho, Moscow ID 83844-1133 USA

4.1 Abstract

Direct and indirect ecological interactions can define the structure of ecosystems and food webs, yet the genetic basis of these interactions is largely unknown for foundation species that are drivers of much larger communities. In a common garden experiment, we tested whether inter- and intraspecific genetic variation in cottonwood (Populus spp.) resistance modulates the indirect effects of a defoliating fungal pathogen (Drepanopeziza populi) on arthropod communities and subsequent herbivory. We found that the pathogen altered arthropod community composition, reduced the richness and abundance of arthropods by as much as 50%, and reduced herbivory by 50%. Interspecific variation in resistance to the pathogen partially modulated arthropod community effects, with heavily defoliated plants strongly affected. In contrast, intraspecific variation in resistance to herbivores modulated the herbivory effect. Pathogens strongly reduced herbivory on the most herbivore-susceptible genotypes, suggesting that pathogen inoculation induced defenses. Our results reveal that pathogens need to be seen not only as agents of plant disease, but also as drivers of cascading effects on associated plant- arthropod interactions. We conclude that the genetic-based interactions of relatively few species can be fundamental to understanding community structure.

4.2 Introduction

The importance of indirect interactions for structuring ecological communities and food webs has long been recognized (1-3). Early work showed that predators suppress the abundance of their plant-eating prey, allowing plants to proliferate and green the world (1). We later learned that herbivores can have far-reaching indirect effects on a broad range of organisms that depend on the same plants by modifying plant structure or inducing resistance (4-8). More recent work has shown that productivity (9) and plant genotypic variation can modulate the indirect effects of herbivores on associated communities and ecosystem processes (10-15). Pathogens too have been implicated in cascading indirect ecological effects (16, 17). By causing disease in foundation species that support much larger communities and ecosystems, pathogens threaten global biodiversity (18, 19). This threat is expected to intensify in future warmer, wetter climates, where pathogens thrive (20). Both positive and negative effects of pathogens on individual herbivores have been reported (21-24). However, compared to our knowledge of the indirect effects of predators and herbivores, we know much less about the nature of pathogen effects (direct or indirect), the dependence of those effects on plant genotypic variation (but see 24, 25), or the response of entire arthropod communities (but see 26). Because plant pathogens generally do not directly attack or kill other organisms dependent on the same plant, such as herbivores, pathogen effects are largely expected to be indirect via their impacts on the host plant (16, 17, 27). Given the importance of plant genetic resistance for determining the severity of pathogen damage (28), indirect effects should be modulated by genetic variation in resistance. Here, we test whether the indirect effects of a defoliating fungal pathogen (Drepanopeziza populi) on foliar arthropod communities and herbivory are modulated by inter- and intraspecific genetic variation in resistance to the pathogen and to herbivory in foundation trees (Populus spp.). Previous work in Populus has shown genetic variation in resistance to this pathogen, and its significant fitness consequences for trees (e.g. 29). We inoculated cuttings of replicated genotypes of two Populus species and their interspecific hybrid (8 genotypes for P. angustifolia, and 4 genotypes each for P.

fremontii and the F1 hybrid P. × hinckleyana) with the pathogen. Disease symptoms were visible on leaves within five days. After inoculation, all treatment and control plants (N=260) were moved into a common garden in Utah containing 20-year-old adult trees of the same taxa, where environmental variation was minimized, and where foliar arthropods were free to colonize and utilize plants. Subsequently, we surveyed foliar arthropod communities on all plants, measured herbivory (percentage of leaf area eaten), and quantified remaining leaf biomass (See Supp Materials and methods). Pathogen severity, in terms of leaf area damaged, varied both among species and their hybrid (treatment by species interaction F=23, p<0.001), and among genotypes within species (Fig 1A&B). Populus angustifolia was the most severely damaged, while P. fremontii and P. × hinckleyana were equally more resistant (Fig 1A&B). Hybrid resistance to the pathogen appears to be inherited from P. fremontii. Pathogen severity, in terms of defoliation, also varied among species and their hybrid (treatment by species interaction F=3.1, p=0.045), and among genotypes within species (Fig 1C&D, Supp Table 1). Similar to leaf area damaged, P. angustifolia was most severely defoliated. P. × hinckleyana was also significantly defoliated, but P. fremontii was not (Fig 1C&D, Supp Table 1). Unlike leaf area damaged, hybrid defoliation in response pathogen infection appears to be inherited from the susceptible species, P. angustifolia. Overall, the two pathogen severity phenotypes – leaf area damaged and defoliation – are correlated (R2=0.38, p<0.001), however the hybrid dissociation in these phenotypes allowed us to discriminate between two hypotheses: 1) pathogen effects on arthropods are modulated by variation in the severity of leaf area damaged, and 2) pathogen effects on arthropods are modulated by variation in the severity of defoliation. In the first scenario, the severity of leaf area damaged would result in stronger arthropod responses (positive or negative) for P. angustifolia, the species with the greatest leaf area infected, and weaker responses for P. fremontii and P. × hinckleyana. Here, a two-way interaction between pathogen treatment and species (or genotype) would predict arthropod responses. In the second scenario, defoliation of susceptible plants would lead to a loss of available habitat for arthropods, and thus a negative effect on arthropods. Here, a three-way interaction between pathogen treatment, species (or genotype) and leaf biomass would predict arthropod responses, with similar and stronger responses for P.

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angustifolia and P. × hinckleyana than for P. fremontii. We also test two alternative hypotheses: 3) pathogen effects on arthropods are modulated by variation in herbivore resistance, and 4) pathogen effects on arthropods are not modulated by plant genetic variation. In the third scenario, the level of herbivore resistance (measured on control plants) would predict arthropod responses. Here, a two-way interaction between pathogen treatment and species (or genotype) would predict arthropod responses. Finally, in the fourth scenario, we would expect similar arthropod responses between Populus species and their hybrid, with the treatment factor predicting effects, but no interaction effects. To evaluate these hypotheses, we used permutational multivariate analysis of variance (PERMANOVA) to estimate variance in arthropod community composition attributable to our predictor variables. And we used restricted maximum likelihood (REML) to estimate variance in richness, abundance and herbivory attributable to our predictor variables (30, 31). For all analyses, we tested the significance of species (genotype nested), pathogen treatment, and their interaction, and the three-way interaction between species (genotype nested), treatment, and remaining leaf biomass. We included stem length as a covariate to account for initial variation in plant size at the start of the experiment. We also evaluated similar models for each Populus species and their hybrid separately to determine whether variation at the genotype level modulates arthropod responses.

4.3 Results and discussion

We observed a total of 32 species of foliar arthropods on plants, representing a total of 11 Orders and 21 Families (Supp Table 2). Of the 32 species, 13 were herbivores (with the highest proportional representation, 41%), 12 predators (37.5%), 4 scavengers (12.5%), and 2 parasites (1 unknown). The pathogen significantly affected the composition of this arthropod community (F=4.4, p<0.001) (Supp Fig 1, Supp Table 3), with the richness (F=22, p<0.001) and abundance (F=29, p<0.001) of arthropods reduced by as much as 50% (Fig 2A&B). Pathogen effects were significant for both herbivores and predators/scavengers (Supp Table 4), although effects were somewhat diluted across

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trophic levels. In accordance with the reduction in herbivore abundance, pathogens reduced herbivory by approximately 50% (F=145, p<0.001) (Fig 3, Supp Table 3). With respect to arthropod community structure, our results support hypothesis 2: interspecific variation in defoliation modulates pathogens’ indirect, negative effects. Arthropods of P. angustifolia and P. × hinckleyana were more strongly affected than the non-defoliated species, P. fremontii (three-way interaction for arthropod richness F=2.1, p=0.059, and abundance F=2.1, p=0.049) (Fig 2A&B, Supp Table 3). Again, these effects were more significant for herbivores than for the entire arthropod community (Supp Table 4). With respect to herbivory, our results support hypothesis 3: intraspecific genetic variation in herbivore resistance modulates pathogens’ indirect, negative effects. Pathogens most strongly reduced herbivory on herbivore-susceptible genotypes, within P. angustifolia only (Fig 3) (treatment by genotype interaction F=2.6, p=0.017). This result suggests that pathogen inoculation induced defense responses that increased resistance to herbivory. Previous studies have found that pathogen infection can induce jasmonate and salicylate responses, which can then negatively affect herbivores (5, 24). While we found evidence that genetic variation in resistance modulates the indirect effects of the pathogen on arthropods, our results also support hypothesis 4. To a large degree, pathogen effects did not depend on plant genetic variation. The pathogen treatment factor alone most strongly predicted arthropod community responses and herbivory (Supp Table 3). This effect was independent of inter- and intraspecific genetic variation (Supp Table 3); even herbivores of P. fremontii, the resistant species, were strongly affected by the pathogen. This result further supports the inference that pathogen inoculation induced herbivore resistance, which negatively and indirectly affected insect herbivores. In summary, we found that the indirect effects of a defoliating pathogen depend, in part, on interspecific genetic variation in defoliation, and intraspecific genetic variation in herbivore resistance. These results support other studies that have found pathogens (native and exotic) affecting foundation plants can lead to significant community and ecosystem consequences (18, 19, 32). We further show that such cascading indirect effects can be genetically determined by resistance to both pathogens and herbivores. We conclude that the interactions of relatively few species (e.g., a plant and a pathogen) can

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define a much larger community of organisms, and that our understanding of community structure needs to incorporate foundation species and their genetic-based interactions.

4.4 Acknowledgements

We thank Alan Le and Denis Willet for their assistance with field work, Roger Guevara for help with data analysis, and Pierre Martineau for help defining arthropod feeding groups. We are grateful for the Ogden Nature Center for hosting this research. This research was supported by the Department of Energy Global Change Environmental Program (PEB), the Heinz Environmental Fellowship (PEB), the Stanford Field Studies Program (PEB), and National Science Foundation Frontiers in Integrative Biological Research grant (DEB-0425908) to the Cottonwood Ecology Group, and an Integrative Graduate Education and Research Traineeship to L.J. Lamit.

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4.6 Figures

Fig 4.1 Intra- and interspecific genetic variation in leaf area damaged (A&B) and resulting defoliation (C&D). Percent leaf area damaged by pathogen inoculation for replicated genotypes within Populus angustifolia, P. fremontii and P. × hinckleyana (A), and means for species and the hybrid (B). Statistically significant differences between Populus species and the hybrid (panel B) indicated by “a” and “b.” Mean remaining leaf biomass for pathogen-inoculated and control plants for replicated genotypes within Populus angustifolia, P. fremontii and P. × hinckleyana (C), and means for species and their hybrid (D). Statistical significance: ** p<0.005.

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Fig 4.2 Mean arthropod richness (A) and abundance (B) with standard error for pathogen-inoculated and control Populus angustifolia, P. fremontii and P. × hinckleyana. Statistical significance: * p<0.05; ** p<0.005.

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Fig 4.3 Herbivory (percent leaf area eaten) on control and pathogen-inoculated replicated genotypes for Populus angustifolia, P. fremontii and P. × hinckleyana. Arrows indicate significant differences in the treatment effect among genotypes of P. angustifolia.

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4.7 Supplementary materials and methods

4.7.1 Tree propagation Twenty replicate dormant cuttings of eight Populus angustifolia genotypes, four P. fremontii genotypes, and four P. × hinckleyana genotypes were collected from mature trees growing in a common garden in Ogden, Utah, in February 2010 (N=320). Common garden trees were originally cloned from randomly selected trees growing in natural stands along the nearby Weber River, and were approximately 19 years-old when cuttings were collected for this study. Cuttings were rooted and raised in book planters containing potting soil at the Northern Arizona University Research Greenhouse, Flagstaff, Arizona. In mid-May 2010, approximately three months after initiating propagation and one month before pathogen inoculation, plants were transferred to 0.8 gallon pots. In mid-June 2010, plants were moved to a greenhouse in Ogden. Mortality reduced our replication to 10-20 plants per genotype, or 5-10 plants per treatment (N=260). Within genotypes, the range of plant sizes was equivalent between pathogen and control treatments.

4.7.2 Pathogen inoculation Drepanopeziza populi is a necrotrophic pathogen that kills host tissue. The pathogen can cause premature defoliation, reduced growth, shoot and branch death, and stem cankers in Populus (33). Damage over multiple years can result in tree death (34). In agreement with these studies, in the study area D. populi negatively affects Populus performance by causing premature leaf abscission (Chapter 3); in outbreak years, susceptible trees can be entirely defoliated by late spring (P. Busby, unpub. data). In the greenhouse in Ogden, pathogen-treatment and control plants were inoculated with asexual spores of the pathogen D. populi suspended in sterilized deionized water and sterile deionized water, respectively. For each P. angustifolia genotype, we inoculated plants with field inoculum collected from adult trees of that genotype growing in the Ogden common garden. Field inoculum for P. fremontii and P. × hinckleyana genotypes was limited, so was augmented with inoculum collected from P. angustifolia genotypes. We moist incubated leaves infected with D. populi for 72 hours

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to initiate sporulation. Leaves were then suspended in sterile deionized water in a closed container and shaken vigorously to dislodge spores. We applied 10ml of inoculum solution or sterile water to leaves of each plant using spray bottles. Plants were covered with plastic bags for 24 hours to maintain moisture at the leaf surface. Lesions developed within five days. Plants remained in the greenhouse for ten days following inoculation, and were then moved into a clearing situated within the center of the Ogden common garden where arthropods were free to colonize and utilize plants. We measured pathogen severity on all plants ten and twenty days after inoculation. We quantified pathogen severity by visually estimating leaf area damaged for three leaves per plant, standardized by leaf age (leaf plastochron index 3, 4, 5). For all leaves, damage was scored on a scale from 0 to 5 reflecting the percentage of leaf area damaged, following the method of Dirzo and Domínguez (1995): 0 = no damage, 1 = 1- 6%, 2 = 7-12%, 3 = 13-25%, 4 = 26-50% or 5 = >50%. Damage scores were then used to calculate a single weighted damage score.

4.7.3 Arthropod and herbivory surveys All plants were placed into the common garden in mid-June and left undisturbed for two weeks before being surveyed for foliar arthropods and herbivory. The timing of study was selected to correspond with peak foliar arthropod activity (35). Whole plant observations (i.e. entire cutting) were performed at each sample time. Observational, non-intrusive arthropod surveys were performed daily for three consecutive days on all replicates of all genotypes. Sampling starting location and order was selected randomly for each sampling day. Arthropods were visually counted and recorded. Unknown arthropods were collected and later identified to species or morphospecies within a family or genera. All arthropods across all sampling times for each replicate were then pooled for total arthropods per replicate. After foliar arthropod surveys were complete, we quantified herbivory for all leaves remaining on each plant using the categorical scale described above, and calculated damage scores (36).

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4.7.4 Plant biomass measurements One week prior to pathogen inoculation, shoot length was measured on all cuttings. Four weeks after pathogen inoculation, remaining leaf biomass was measured destructively to determine the effect of the pathogen on the leaf resource available to arthropods. Leaves were oven dried at 65ºC for at least 3 days before masses were obtained.

4.7.5 Statistical analyses All statistical analyses were conducted in R 2.8.1 (R Development Core Team 2008). First, we used restricted maximum likelihood (REML) analysis to test how plant species, genotype (nested within species), pathogen treatment, and their interactions affect remaining leaf biomass. Next, we tested how the pathogen affects arthropod community structure and herbivory. We used permutational multivariate analysis of variance (PERMANOVA, 37) to test if pathogen inoculation affects arthropod community composition. Our community matrix consisted of columns of arthropod species’ abundances, each species divided by the maximum value observed for that species to down-weight effects of extremely abundant species in order to ensure that they do not drive patterns perceived as community wide effects. To visualize community results, we utilized two-dimensional representations of communities where the x and y-coordinates are based on non-metric multidimensional scaling (NMDS) analysis (isoMDS, vegan package). We used restricted maximum likelihood (REML) analysis to test if pathogen inoculation affects arthropod richness (log-transformed), abundance (log-transformed) and herbivory (30, 31). For all analyses (biomass, arthropod community structure, and herbivory), we evaluated models for each Populus species and their hybrid separately to determine whether intraspecific variation modulates responses. In addition, for arthropod community structure analyses, we evaluated herbivores and non-herbivores (predators/scavengers) separately to test if the pathogen effect is diluted across trophic levels. For all PERMANOVA and REML models, we tested the significance of species (genotype nested), pathogen treatment, and their interaction, and the three-way interaction between species (genotype nested), treatment, and remaining leaf biomass.

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We included stem length as a covariate to account for variation in plant size. For all analyses, genotype was a random effect; all other factors were fixed. For PERMANOVA, the significance of each factor was tested used F-tests based on sequential sums of squares from permutations of the raw data (37). For REML, the significance of genotype was tested using log-likelihood ratio tests (30). The significance of fixed effects was tested using F-tests (37).

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4.8 Supplementary tables

Table 4.S1 Model results showing the significance of predictor variables for leaf biomass. P. Full model angustifolia P. fremontii P. x hinckleyana F* p F* p F* p F* p Leaf biomass Species 1.8 0.16 Treatment 55 <0.001 88 <0.001 0.88 0.35 25 <0.001 Species X Treatment 3.1 0.045 Stem Length 155 <0.001 95 <0.001 72 <0.001 56 <0.001 Genotype 9.8 <0.001 12 <0.001 33 <0.001 Genotype X Treatment 5.2 <0.001 2.4 0.074 3.3 0.027 *χ2 for Genotype

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Table 4.S2 Foliar arthropods encountered in surveys, and their feeding habit. Feeding Arthropod Order Family Genus Species Habit Anthocoreid Hemiptera Anthocoreidae Anthocoris antevolens Predaceous BEB Hemiptera Rhopalidae Boisea trivitatta Herbivorous

Braconid Hymenoptera Braconidae 1 Parasitoid Leafhopper (brn mttld) Hemiptera Cicadellidae 2 Herbivorous Cercopid (brn) Hemiptera Cercopidae 1 Herbivorous Chait. (grn) Hemiptera Aphididae Chaitophorus populellus Herbivorous

Coen. Odonata Coenagrionidae Argia 1 Predaceous Chrys. egg Neuroptera Chrysopidae Chrysopa 1 Predaceous fly (dolich.) Diptera Dolichopodidae 1 Predaceous fly (muscid) Diptera Muscidae 1 Scavenger fly (unkn.) Diptera 1 unknown grasshppr Orthoptera Acrididae Melanoplus sanguinipes Herbivorous Leafhopper (fisheye) Hemiptera Cicadellidae 3 Herbivorous Leafhopper (grn) Hemiptera Cicadellidae Gypona 4 Herbivorous Leafhopper (skunk) Hemiptera Cicadellidae 1 Herbivorous Leafhopper (turq.) Hemiptera Cicadellidae 5 Herbivorous Membracid (grn) Hemiptera Membracidae 1 Herbivorous midge Diptera Chironomidae 1 Herbivorous mite (red) Acari Trombiculidae 1 Parasitic ant (myrmecine) Hymenoptera Formicidae Tetramorium caespitum Scavenger Cocc. Nymph Coleoptera Coccinellidae 1 Predaceous punky Diptera Ceratapogonidae 1 Predaceous spider (Araniella) Araneae Araneidae Araniella displicata Predaceous spider (lycosid) Araneae Lycosidae 1 Predaceous spider (unkn. Purp.) Araneae Araneidae 1 Predaceous spider (unkn. tan/gry) Araneae Araneidae 2 Predaceous stem borer Lepidoptera Tortricidae Gypsonoma haimbachiana Herbivorous thrip (blk) Thysanoptera Thripidae Phlaeothripidae 1 Predaceous thrip (yllw) Thysanoptera Thripidae Phlaeothripidae 2 Predaceous T-dip Diptera Muscidae 2 Scavenger tiny T-dip Diptera Muscidae 3 Scavenger weevil (blk) Coleoptera Curculionidae Anthonominae 1 Herbivorous

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Table 4.S3 PERMANOVA and REML results showing the significance of predictor variables for arthropod community composition, abundance, richness, and herbivory.

Full model P. angustifolia P. fremontii P. x hinckleyana Community F* p F* p F* p F* p Treatment 4.4 <0.001 2.1 0.017 2.1 0.021 2.7 0.006 Species 1.4 0.055 Treatment X Species 1.03 0.41 Treatment X Species X leaf biomass 1.1 0.28 Stem length 0.76 68 0.85 0.6 0.96 0.47 1.4 0.18 Genotype 0.76 0.91 0.85 0.704 1.1 0.37 Treatment X Genotype 1.1 0.27 1.4 0.063 0.71 0.902 Treatment X Genotype X leaf biomass 0.76 0.91 1.9 0.01 1.1 0.26

Arthropod abundance Treatment 29.9 <0.001 9.6 0.0026 4.7 0.035 11 0.0015 Species 1.6 0.21 Treatment X Species 0.17 0.85 Treatment X Species X leaf biomass 2.1 0.049 Stem length 10 0.0015 2.4 0.12 1.8 0.19 0.72 0.4 Genotype 0.15 0.99 0.3 0.82 0.17 0.91 Treatment X Genotype 1.5 0.18 1.4 0.24 0.45 0.72 Treatment X Genotype X leaf biomass 0.96 0.51 1.2 0.34 1.6 0.14

Arthropod richness Treatment 24.2 <0.001 13.2 <0.001 5.5 0.0023 11 0.0016 Species 1.3 0.26 Treatment X Species 0.41 0.66 Treatment X Species X leaf biomass 2.1 0.059 Stem length 8.7 0.0036 1.9 0.17 0.87 0.35 1.2 0.27 Genotype 0.23 0.96 0.45 0.72 0.37 0.78 Treatment X Genotype 1.3 0.26 1.7 0.18 0.56 0.64 Treatment X Genotype X leaf biomass 0.93 0.54 1 0.45 1.4 0.22

Herbivory Treatment 145 <0.001 69 <0.001 48 <0.001 42 <0.001 Species 5.1 0.00704 Treatment X Species 0.31 0.73 Stem length 3.3 0.072 Genotype 0.5 0.83 0.32 0.81 0.505 0.68 Treatment X Genotype 2.6 0.017 1.5 0.23 0.601 0.62 *χ2 for Genotype in abundance, richness and herbivory analyses

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Table 4.S4 Model results showing the significance of predictor variables for herbivore and predator community composition, abundance and richness.

Herbivores Predators F p F p Arthropod abundance Treatment 14 <0.001 11 0.0011 Species 0.87 0.42 0.39 0.68 Treatment X Species 0.81 0.44 0.049 0.95 Treatment X Species X leaf biomass 2.5 0.023 1.04 0.4 Stem length 3.8 0.051 0.86 0.35

Arthropod richness Treatment 18 <0.001 14 <0.001 Species 0.7 0.5 0.51 0.6 Treatment X Species 0.47 0.63 0.08 0.92 Treatment X Species X leaf biomass 2.2 0.042 1.2 0.32 Stem length 5.1 0.025 1.8 0.18

Community Treatment 4.1 0.002 1.7 0.11 Species 1.8 0.02 0.54 0.91 Treatment X Species 1.1 0.36 0.14 0.13 Treatment X Species X leaf biomass 1.01 0.49 1.1 0.24 Stem length 1.2 0.31 0.47 0.87

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4.9 Supplementary figures

Fig 4.S1 Two-dimensional representation of arthropod communities found on pathogen- inoculated and control plants for eight P. angustifolia genotypes, four P. fremontii genotypes and four P. × hinckleyana genotypes. Coordinates are based on global, non- metric multidimensional scaling (NMDS) analysis. Error lines are standard error of the mean of the ordination axes scores.

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