Arbuscular mycorrhizal fungi (AMF) play an important role in nutrient cycling and growth of flax (Linum usitatissimum L.). However, limited information is available regarding the symbiotic association between flax and AMF in field environments. A study was conducted to survey AMF communities colonizing flax grown in Saskatchewan. Additionally, field and growth chamber studies investigated the impact of AMF inoculation on nutrient uptake and growth of flax. Eighteen commercial flax fields were surveyed to assess mycorrhizal colonization of flax and to assess the impact of agricultural practices and soil abiotic factors on AMF activity. The flax root-associated AMF communities were explored using a 454 sequencing method, together with microscopic-based measurements of root AMF colonization and soil spore density. High levels of root colonization were detected in most flax fields. Of the 222 AMF operational taxonomic units (OTUs) identified in flax roots, 181 OTUs clustered as Funneliformis-Rhizophagus, 19 as Claroideoglomus, 14 as Paraglomus, six as Diversisporales and two as Archaeospora. Results suggest that tillage influenced the composition of AMF communities colonizing flax, and reduced relative AMF abundance and species richness. Additionally, AMF community characteristics were related to soil abiotic factors such as pH, EC, available phosphorus and nitrogen. Field experiments were conducted over two years (two sites per year) using a commercial AMF inoculant applied at three rates (0, 1X, and 2X the recommended rate) with or without P fertilizer (16.8 kg ha-1). The response of flax cultivars to AMF inoculation was examined in a growth chamber experiment. In addition, 454 sequencing was employed to examine the impact of AMF inoculation on root-associated AMF communities. Under field conditions, only one site showed increased root colonization with AMF inoculation. Flax responded to AMF inoculation differently under different field conditions. At the two sites with intermediate initial soil P level, evidence of increased above-ground biomass and plant nutrient uptake with AMF inoculation was observed. However, such an effect was not detected when P fertilizer was combined with the inoculation. At a low P site and an irrigated site, P application accounted for all of the increases in plant nutrient uptake and biomass of flax, whereas no responses to AMF inoculation were detected. The 454 sequencing revealed different inoculation-induced changes in the diversity and composition of root-associated AMF communities between sites, which was possibly related to different field environments and native AMF communities. In the growth chamber, AMF inoculation resulted in general increases of plant nutrient uptake among cultivars, but only one cultivar showed enhanced biomass with inoculation. The diversity of AMF communities colonizing different flax cultivars was generally reduced by AMF inoculation. Community composition shifted under AMF inoculation, and the shifts appeared to be cultivar specific. These results suggested that benefits of AMF inoculation in flax production are limited and currently not predictable, and the degree of response is likely dependent on a myriad of soil and environmental conditions.

Doctor of Philosophy / Division of Biology / Ari M. Jumpponen / Microorganisms are critically important for establishing and maintaining ecosystem properties and processes that fuel and sustain higher-trophic levels. Despite the universal importance of microbes, we know relatively little about the rules and processes that dictate how microbial communities establish and assemble. Largely, we rely on assumptions that microbial community establishment follow similar trajectories as plants, but on a smaller scale. However, these assumptions have been rarely validated and when validation has been attempted, the plant-based theoretical models apply poorly to microbial communities. Here, I utilized genomics-inspired tools to interrogate microbial communities at levels near community saturation to elucidate the rules and patterns of microbial community assembly. I relied on a community filtering model as a framework: potential members of the microbial community are filtered through environmental and/or biotic filters that control which taxa can establish, persist, and coexist. Additionally, I addressed whether two different microbial groups (fungi and bacteria) share similar assembly patterns. Similar dispersal capabilities and mechanisms are thought to result in similar community assembly rules for fungi and bacteria. I queried fungal and bacterial communities along a deglaciated primary successional chronosequence to determine microbial successional dynamics and to determine if fungal and bacterial assemblies are similar or follow trajectories similar to plants. These experiments demonstrate that not only do microbial community assembly dynamics not follow plant-based models of succession, but also that fungal and bacterial community assembly dynamics are distinct. We can no longer assume that because fungi and bacteria share small propagule sizes they follow similar trends. Further, additional studies targeting biotic filters (here, snow algae) suggest strong controls during community assembly, possibly because of fungal predation of the algae or because of fungal utilization of algal exudates. Finally, I examined various technical aspects of sequence-based ecological investigations. These studies aimed to improve microbial community data reliability and analyses.

A major goal of ecology is to understand the factors that shape interactions among species. In this study, I explored the little-known associations between beetles and macrofungal fruiting bodies to characterize patterns of beetle-fungus association and to investigate sources of variation in the structure of these trophic interactions. First, I characterized the composition and diversity of beetle-sporocarp associations at two sites in the Appalachian Mountains and foothills, and evaluated the extent to which beetle community structure varied with fungal species, sporocarp age, and sporocarp dry mass. My results showed that beetle abundance and diversity differed among fungal species and were positively associated with sporocarp age and dry mass. I also found evidence of a nested structure in beetle-sporocarp interactions, wherein specialists on both sides of the association interact preferentially with more generalized species. Next, I performed a field study of beetle-sporocarp associations over two summers to evaluate the factors related to interaction breadth in trophic associations. I found evidence that interaction breadth varies with the palatability of the food organism (as indicated by sporocarp toughness and sporocarp age) and showed that beetle interaction breadth was negatively correlated with sporocarp persistence. I found strong intraseasonal variation in interaction breadth, but no evidence that this variation was structured by precipitation or differences in beetle community composition. In my third chapter, I conducted a field experiment to investigate (1) the importance of an individual food organism's physical properties in determining its relative importance in the beetle-sporocarp interaction network and (2) whether the structure of the beetle-sporocarp interaction network cycles predictably with the time of day. My results show that size and density of individual food organisms may be important factors in determining their relative importance in an interaction network, and offer the first evidence of diurnal cycling in the structure of interaction networks.

The biochemistry of leaves merges the fates of trees and the atmosphere. Leaf primary metabolism cycles carbon and indirectly drives atmospheric circulation via the latent heat of transpiration. Tropical forests contain half of global forest carbon, and actively cycle carbon and energy year round, making them critical components of the coupled biosphere-climate system. Climate change threatens tropical forests with rising temperatures and increasing variability of precipitation. Their response will influence future biodiversity as well as the fate of the climate. Understanding the physiological attributes that define tropical tree responses and feedbacks to climate is a current research priority. The emission of isoprene gas from plant leaves has been demonstrated to enhance leaf tolerance to high temperatures and drought. Isoprene is a volatile secondary metabolite produced in the chloroplast by approximately one-third of plant species. While the benefits of isoprene are supported by extensive laboratory and greenhouse-based research, work has only begun to explore how the trait is integrated in plant functional strategies. Whether isoprene influences differential species performance and survival across environments has yet to be tested. An impediment to filling this clear ecological research gap has been a lack of instrumentation capable of quantifying isoprene emissions from leaves in remote field settings. The first study presented here tests the hypothesis that isoprene emission influences plant community assembly shifts across environmental gradients and through time in tropical forests. The capacity for a species to produce isoprene was associated with increased relative abundance at higher temperatures and following drought anomalies. A negative relationship with the length of seasonal drought suggests a trade-off between isoprene emission and other plant traits, such as deciduous leaf habit. The second study presents the development of a new instrument that is uniquely optimized for field-based ecological research on leaf volatiles. The new system, named PORCO (Photoionization of Organic Compounds), utilizes custom leaf cuvettes, precision light control, and an optimized commercial photoionization detector to achieve real-time detection of leaf emissions with detection limits better than 0.5 nmol m⁻² leaf s⁻¹. The third study utilizes PORCO to test hypotheses about the structuring of isoprene within plant functional strategies and across forest microenvironments in an eastern Amazonian evergreen tropical forest. The results support the role of isoprene—and potentially other volatile isoprenoids—in mitigating effects of intermittent sun exposure in the sub-canopy. Emissions are structured in a complex, multivariate manner that depends on taxonomy, leaf and wood characteristics, tree height, and light environment. The results from this dissertation work demonstrate that isoprene emission from leaves affects plant responses to climate at ecologically relevant scales. Isoprene influences climate not only by its effect on primary leaf functions, but also by directly altering atmospheric chemistry, and contributing to aerosol and cloud properties. Understanding isoprene's role in forest responses to increasing temperatures and drought will help to predict the feedbacks between forest ecosystems and climatic change.

Plants can be colonized by complex communities of endophytic fungi. This thesis presents two studies, both of which investigate biotic filtering in endophytic fungal communities. Chapter 1. Endophytic fungi can be acquired horizontally via propagules produced in the environment such as in plant litters of various species. Given that litters from different plant species harbor distinct endophytic fungal communities and that endophytic fungi may be dispersal-limited, the structure of the endophytic fungal community of a given plant may be determined by proximity to particular inoculum sources. Community assembly may also be affected by biotic filtering by the plant. Therefore, a plant may be able to select particular fungal taxa from among the available pool. In that case, the structure of the endophytic fungal community in the plant could be somewhat independent of the structure of the inoculum community. We tested the hypothesis that biotic filtering of endophytic fungal communities occurs in Bromus tectorum by exposing it to a variety of inoculum sources including litters from several co-occurring plant species. The inoculum sources differed significantly from each other in the structures of the communities of endophytic fungi they harbored. We characterized the structures of the resulting leaf and root endophytic fungal communities in Bromus tectorum using high-throughput sequencing. All tested inoculum sources successfully produced complex communities of endophytic fungi in Bromus tectorum. There was significantly more variation in the structures of the communities of endophytic fungi among the inoculum sources than in the resultant endophytic fungal communities in the leaves and roots of Bromus tectorum. These results suggest that biotic filtering by Bromus tectorum played a significant role in the assembly of the endophytic fungal communities in tissues of Bromus tectorum. Because endophytic fungi influence plant fitness, it is reasonable to expect there to be selective pressure to develop a uniform, desirable endophytic fungal community even from disparate inoculum sources via a process known as biotic filtering. Chapter 2. Frequently one finds that different plant species harbor communities that are distinct. However, the nature of this interspecific variation is not clear. We characterized the endophytic fungal communities in six plant species from the eastern Great Basin in central Utah. Four of the species are arbuscular mycorrhizal (two in the Poaceae and two in the Asteraceae), while the other two species are nonmycorrhizal (one in the Brassicaceae and one in the Amaranthaceae). Our evidence suggests that both host mycorrhizal status and phylogenic relatedness independently influence endophytic fungal community structure.

At broad scales, community ecologists study how biogeographic factors like environmental dissimilarity and geographic distance influence community assembly and composition. At small scales, community ecologists study how one or several species interact to determine habitat partitioning and coexistence. In this dissertation, I present studies at both scales. Chapter One investigates community assembly across the Caribbean, Central, and South American radiations of Anolis lizards and Eleutherodactylid frogs to test whether oceanic islands are unique in their assembly processes. Such uniqueness is suggested by high levels of endemism on islands; however, comparable levels of endemism can be found in mainland communities. I modeled the rate of species turnover between mainland communities, with respect to geographic distance and environmental dissimilarity, and then used the mainland model to predict turnover among islands. Turnover among island communities was significantly higher than predicted from the mainland model, confirming the long-held but untested assumption that island assemblages accumulate biodiversity differently than their mainland counterparts. Chapter Two reviews the evidence for ecological character displacement (ECD), an evolutionary process whereby two resource competitors diverge from one another in phenotype and resource use, facilitating coexistence in a community. I find that, despite current scientific opinion, the evidence for ECD is equivocal; most cases of ECD pattern fail to rule out processes alternative to resource competition that could create the same pattern. I conclude that better evidence may come from real time tests of ECD. Chapters Three and Four describe just such a test in small island populations of Anolis carolinensis. In Chapter Three, I find that small island populations of A. carolinensis that have come into sympatry with a novel competitor, the invasive A. sagrei, shift their habitat use to become more arboreal, compared to allopatric populations. Consistent with prediction, individuals from sympatric populations have larger toepads with additional adhesive scales - a common adaptation to arboreality in Anolis. In Chapter Four, I describe a common garden experiment that finds that the observed toepad divergence is an evolved response, suggesting rates of divergence for toepad area and scale number on par with well known examples of contemporary evolution.

Infection by multiple parasites is a part of everyday life for many organisms. The host immune system may be a central mediator of the many ways parasites might influence one another (and their hosts). Immunity provides a means for the colonized to reduce the success of current and future colonizers and has evolved across the tree of life several times independently. Along the way, the immune systems of plants as well as many groups of animals has evolved perhaps an accidental vulnerability wherein defense against one parasite can increase susceptibility to others. This so-called immune 'cross-talk' is a conundrum worth investigating not only to understand the impact of parasites on focal organisms, but also to better predict how immunity itself influences the evolution and epidemiology of parasites whose spread we might like to curtail. For plants, co-infection often comes from insect herbivores and various bacteria that colonize the leaf interior. Both colonizers can reduce plant fitness directly or indirectly by potentiating future enemies via cross-talk in plant immunity. This phenomenon has largely been studied in laboratory model plants, leaving a substantial gap in our knowledge from native species that interact in the wild. This dissertation helps close this gap by investigating the ecology of co-infection of a native plant by its major insect herbivore and diverse leaf-colonizing bacteria. I revealed that leaf co-infection in the field by leaf-mining herbivores and leaf-colonizing ("phyllosphere") bacteria is substantially more common than single infection by either group and that bacterial infection can cause increased feeding by herbivores in the laboratory. Immune cross-talk can also shape the field-scale patterns of herbivory across a native plant population. Studying the main herbivore of this native plant in detail revealed that, in contrast to many specialist herbivores, our focal species avoids plant defenses likely because it does not possess a specialized means of avoiding their toxicity. Nonetheless, this species may depend on the very same defenses it avoids by being initially attracted to plants that produce them. This foraging strategy is unique among known specialists. Lastly, I moved beyond immune cross-talk to explore how co-occurring phyllosphere bacteria might directly impact one another through competition. In the lab, I found that different growth strategies underlie competitive ability for two major clades of bacteria within the genus Pseudomonas, and that toxin production and resistance may be important mediators of competition within the phyllosphere. However, competitively superior bacteria that produce toxins may indirectly facilitate the survival of inferior competitors through their being toxin resistant, which likely enhances co-existence of diverse bacteria in the phyllosphere. Together, this dissertation has revealed a variety of means by which co-infecting bacteria and insects might influence one another through plant defense cross-talk, as well as how the complex interplay of colonization and competition might affect the structure of leaf microbial communities in nature.

This dissertation presents four microbial microcosm-based experimental studies addressing questions related to the causes and consequences of biodiversity. All four studies adopted an approach that integrates ecology and evolutionary biology. Two studies explored the utility of knowledge on species phylogenetic relationships for understanding community assembly (chapter 1) and invasibility (chapter 3). The other two studies investigated the impacts of important ecological factors, including competition (chapter 2) and temporal niches (chapter 4), on adaptive radiation, using the rapidly diversifying bacterium Pseudomonas fluorescens SBW25 as the model organism. The first study, described in Chapter 1, examined how phylogenetic relatedness between competing species affected the strength of priority effects and ecosystem functioning during community assembly. Strong priority effects emerged only when competing bacterial species were phylogenetically most closely related, resulting in multiple community states associated with different assembly histories. In addition, the phylogenetic diversity of bacterial communities effectively predicted bacterial production and decomposition. The second study, described in Chapter 2, explored the role of competition in the adaptive radiation of P. fluorescens. The adaptive radiation was generally suppressed by competition, but its effect was strongly modulated by the phylogenetic relatedness between the diversifying and competing species and their immigration history. The inhibitive effect of competition on adaptive radiation was strongest when phylogenetic relatedness was high and when competitors were introduced earlier. The third study, described in Chapter 3, evaluated the relative importance of phylogenetic relatedness between resident and invading species and phylogenetic diversity of resident communities for invasibility. Laboratory bacterial communities containing a constant number of resident species with varying phylogenetic diversity and relatedness to invaders were challenged by nonresident bacterial species. Whereas invader abundance decreased as phylogenetic relatedness increased as predicted by Darwin's naturalization hypothesis, it was unaffected by phylogenetic diversity. The final study, described in Chapter 4, presented the first experimental demonstration of the maintenance of biodiversity that emerged from adaptive radiation in the presence of temporal niches. Only when provided with temporal niche opportunities were multiple derived phenotypes of P. fluorescens able to coexist as a result of negative frequency-dependent selection. When temporal niche was absent, the specialized phenotypes either did not emerge or were predominated by one superior phenotype.

Traditionally, the study of biodiversity has focused on quantifying patterns of species diversity, or species richness, by simply counting the number of species across environmental gradients. This approach has been fundamental to ecological investigations and thinking with regards to identifying patterns of biodiversity. Although species diversity is the most commonly used dimension of biodiversity, species diversity alone does not provide a mechanistic understanding of biodiversity gradients. By also quantifying the genetic and phylogenetic diversity of a population, community or ecosystem, ecologists can become more informed on the relationships organisms have with one another, as well as their potential to adapt to changes in their environment. While each of these approaches provides methods for characterizing biodiversity, they do not offer direct insight into what species do, how they function, or how they will respond to changes in their environment. Functional, or trait-based ecology, provides an informative alternative to species-centric approaches that seeks to understand patterns of biodiversity in terms of functional traits. Functional traits capture fundamental tradeoffs in life history strategies that can be used to determine species ecological roles and can be used to scale from organisms to ecosystems to ask broad ecological questions. The overarching goal of my dissertation is to add additional links to trait-based ecology by identifying potential sources of trait variation across different spatial and temporal gradients between varying levels of biological organization. By assessing variation across spatial-temporal scales, I tested two prominent assumptions of trait-based ecology. First, I tested the trait-environment assumption wherein traits affect ecosystem processes. Therefore, there should be a predictable relationship between traits, their environment, and ecosystem function across large ecological gradients and between broad taxonomic groups. Second, I tested the assumption that interspecific trait variation exceeds intraspecific trait variation; thus, the species mean trait value captures much of the variation for a given trait. My study systems include the latitudinal diversity gradient of North America, forests of varying successional age in the tropical dry forests of Costa Rica, and a subalpine meadow of Colorado. First, we collected leaf trait data and soil microbial data at six sites across the latitudinal diversity gradient to test a central hypothesis of trait-based ecology, primarily that shifts in plant traits associated with decomposition and nutrient availability ramify to influence microbial functioning. We found that changes in plant traits not only reflect nutrient limitation across broad ecological gradients, but also have important regional effects on biogeochemical processes, microclimates, and energy fluxes that influence microbial diversity. Furthermore, changes in plant function correspond to changes in bacterial functional traits related to carbon, nitrogen, and phosphorus cycling, although only fungal functional traits related to nitrogen cycling change across the gradient. Our results represent one of the first comparisons of functional diversity within and across bacterial, fungal, and plant communities across a latitudinal gradient. Next, we collected leaf functional trait and abiotic data across a 110-year chronosequence within a tropical dry forest in Costa Rica. We focused on six leaf functional traits for woody plants within 14 plots that have varying times since disturbance in the tropical dry forests of Guanacaste, Costa Rica. When we compare species composition and community function, we find that older tropical dry forest communities differ significantly from younger forests in species composition, above ground biomass, and functional traits. Species in younger forests have traits better adapted to hotter temperatures and increased drought. For example, young forests are characterized by thicker leaves with higher water use efficiency. In contrast, older forests have thinner broader leaves more susceptible to desiccation. Interestingly, in contrast to expectations, variation in these functional traits does not generally change through succession. This means that the different species within each community are converging on similar functional strategies. Our results also suggest that regenerating tropical dry forests are resilient and can be restored within a human lifetime. Finally, we evaluated patterns of trait variation within and between three years to understand the widely-ignored source of temporal variation associated with seasonality and test the assumption that interspecific trait variation exceeds intraspecific variation and the species means account for the overall variation of a trait. To do this, we collected leaf data from eight species at a local site in Colorado throughout the growing season, over three years, as well as extracted data from a global database and made comparisons to assess sources trait variation. We found that, although the timing of collection influences one’s ability to capture fine-scale processes occurring on short time scales, collecting data locally throughout the growing season and across multiple years does not significantly influence species ranking. However, species ranking is not conserved for comparisons between local and global databases. This suggests that extra care should be taken when applying global data for species-specific studies and that ‘snap-shot’ sampling designs may over- or underestimate community trait distributions, reducing predictability. Overall, this body of work extends beyond understanding patterns of species diversity through the inclusion of species function. It contributes to our understanding of variation in biodiversity across broad ecological gradients and between diverse taxonomic groups, how communities assemble via functional traits, and the importance of temporal variation on functional traits for detecting fine-scale patterns.

Seascape ecology provides a useful framework from which to understand the processes governing spatial variability in ecological patterns. Seascape context, or the composition and pattern of habitat surrounding a focal patch, has the potential to impact resource availability, predator-prey interactions, and connectivity with other habitats. For my dissertation research, I combined a variety of approaches to examine how habitat quality for fishes is influenced by a diverse range of seascape factors in sub-tropical, back-reef ecosystems. In the first part of my dissertation, I examined how seascape context can affect reef fish communities on an experimental array of artificial reefs created in various seascape contexts in Abaco, Bahamas. I found that the amount of seagrass at large spatial scales was an important predictor of community assembly on these reefs. Additionally, seascape context had differing effects on various aspects of habitat quality for the most common reef species, White grunt Haemulon plumierii. The amount of seagrass at large spatial scales had positive effects on fish abundance and secondary production, but not on metrics of condition and growth. The second part of my dissertation focused on how foraging conditions for fish varied across a linear seascape gradient in the Loxahatchee River estuary in Florida, USA. Gray snapper, Lutjanus griseus, traded food quality for quantity along this estuarine gradient, maintaining similar growth rates and condition among sites. Additional work focused on identifying major energy flow pathways to two consumers in oyster-reef food webs in the Loxahatchee. Algal and microphytobenthos resource pools supported most of the production to these consumers, and body size for one of the consumers mediated food web linkages with surrounding mangrove habitats. All of these studies examined a different facet of the importance of seascape context in governing ecological processes occurring in focal habitats and underscore the role of connectivity among habitats in back-reef systems. The results suggest that management approaches consider the surrounding seascape when prioritizing areas for conservation or attempting to understand the impacts of seascape change on focal habitat patches. For this reason, spatially-based management approaches are recommended to most effectively manage back-reef systems.