Intimate, mutualistic, association with microbes is a common mechanism for organisms to utilize certain niches. Insects are a particularly well-studied group in this respect, frequently forming long-term, obligate associations with symbiotic microbes. These symbioses are often nutritional in nature, with the symbiont providing the host with nutrients that are otherwise unavailable. Aphids are notable for their well-defined relationship with the symbiotic Bacteria Buchnera aphidicola. By synthesizing the amino acids the aphid is unable to produce itself, Buchnera permits its host to feed on plant phloem, which lacks sufficient quantities of these essential nutrients. Buchnera, as with many obligate intracellular symbionts, has a reduced effective population size (Nₑ) due to asexual reproduction and severe population bottlenecks experienced during transmission between generations. The reduction in Nₑ has facilitated the degradation of the symbiont genome through fixation of deleterious mutations via drift. The consequences of accelerated evolutionary rates has been examined primarily through genome sequencing and comparative studies of symbionts from different host species. The work detailed in this dissertation examines the role of deleterious mutations and drift at multiple taxonomic levels. Analysis of aphid amino acid requirements utilizing an artificial diet assay revealed variation in clones of the pea aphid, Acyrthosiphon pisum. In one clone, a mutation in the arginine biosynthesis pathway appears to underlie a host dietary requirement for arginine. Examination of the number of Buchnera within an A. pisum clone also revealed variation in symbiont titer between clones. When compared across F₁ offspring of cross between a low- and a high-titer clone, extensive variation was observed in titer that exceeded variation observed in field-collected clones. No maternal effects were observed, suggesting that Buchnera is not in control of its replication. At a broader taxonomic scale, the replacement of Buchnera in the aphid Cerataphis brasiliensis was examined by sequencing the genome of its fungal symbiont (YLS). The genome of the YLS revealed a much greater metabolic capacity than Buchnera, possibly due to its extracellular habitat. The YLS exhibited signatures of elevated evolutionary rates and intron gain consistent with a reduction in Nₑ due to its symbiotic niche.

Drought-induced tree mortality is an emerging global phenomenon that appears related to climate change and rising temperatures in particular, and may be an early indication of vegetation change. However, vegetation response to climate change is uncertain, particularly for future novel climates. Notably, no current models of vegetation change attempt to mechanistically predict plant mortality, and in particular, mortality of trees, which exerts strong influences on ecological function. Resolving uncertainties surrounding the physiological mechanism and temperatures sensitivity of tree mortality is a current challenge in global change ecology. The objectives of this dissertation were to 1) consider tree mortality consequences for earth system processes related to carbon, water, and energy exchange that include climate regulation; 2) explore tree mortality effects on the water cycle by developing hypotheses and research needs; 3) quantify the temperature sensitivity of drought-induced tree mortality and gain insight into the physiological mechanism of mortality; 4) quantify the relationships among temperature, stored carbohydrate resources, and gas exchange to further elucidate physiological tree mortality mechanisms; and 5) quantify the sensitivity of two species of pine seedlings to progressively elevated temperatures and relate mortality to the effect of temperature on carbon metabolism. Major findings of this dissertation relate to the temperature sensitivity, physiological mechanism, and implications of tree mortality. Assessment of the potential consequences of tree mortality for earth system processes documented the contrasting influences of tree mortality on the terrestrial C cycle and land-surface energy exchange, the balance of which will determine the net effects on climate regulation (Appendix A). Following a survey of the ecohydrology literature, thresholds for tree mortality to cause watershed changes were hypothesized at ~20% loss of canopy cover, ~500 mm of annual precipitation, and whether flows are snowmelt dominated (Appendix B). Elevated temperature (~+4°C) accelerated tree mortality by 28% during experimental drought, a difference related to cumulative respiration dynamics in piñon pine (Appendix C). Stored carbohydrate resources were declined during lethal drought but were not entirely depleted prior to mortality (Appendix D). Seedlings exhibited progressive declines in time-to mortality with increased temperatures, a response related to C metabolism (Appendix E).

Subalpine ecosystems are experiencing rapid changes in snow pack, temperature, and precipitation regime as a result of anthropogenic climate forcing. These changes in climate can have a profound effect on subalpine ecosystem structure and functioning, which may ultimately feed back to climate change. In this study, I examined the response of the subalpine meadow plant communities at the Rocky Mountain Biological Laboratory to natural and simulated climate change. First, I looked at whether changes in growing season precipitation or temperature regime would have the larger effect on subalpine ecosystem carbon flux. In a simulated warming experiment, changes in growing season precipitation had a tenfold larger effect on cumulative carbon flux than did the warming treatment. Along a natural climatic and elevational gradient, precipitation stimulates carbon uptake, particularly at higher elevations. Given projected decreases in summer precipitation in the high elevation Rockies, we predict a 20% decrease in carbon uptake from subalpine meadows. Second, I compared the taxonomic, phylogenetic and functional structure of plant communities along an elevational gradient to infer which climatic and biotic factors influence community assembly at each elevation. Floral and phenology traits become overdispersed at high elevation, mirroring phylogenetic relatedness, and suggesting pressure to diversify to attract pollinators during the abbreviated growing season. At the same time, leaf functional traits become clustered at high elevation, indicating multiple opposing assembly mechanisms in subalpine communities. Finally, I studied the natural history of sagebrush, Artemisia tridentate ssp. vaseyana, at its elevational range limit in subalpine meadows. In particular, I focused on the importance of warming and species interactions in elevational advance of the species. I found that facilitation by neighboring forbs was critical for sagebrush seedling survival, decreasing mortality by 75%. Seedling mortality was overwhelmingly due to desiccation of seedlings; therefore, neighboring forbs moderate temperature and water stress for seedlings. Despite the extremely limited growing season at high elevation caused by subfreezing temperatures, subalpine ecosystem structure and function are closely tied to water availability during the growing season. Therefore, improved predictions of future precipitation regimes over the Rocky Mountains will be our best tool for conservation of these fragile habitats.

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.

Viruses are the most abundant biological entity on Earth and outnumber their hosts ten-to-one. Ocean viruses (phages) impact bacterial-driven global biogeochemical cycles through lysis, manipulating host metabolism, and horizontal gene transfer. However, knowledge of virus-host interactions and viral roles in ecosystems remains limited due to few cultured marine phage genomes and non-quantitative culture-independent metagenomes. Here, I develop and apply novel and well-tested bioinformatic techniques to explore Pacific Ocean viral communities using quantitative datasets derived from rigorously-tested preparation methods. To evaluate concentration and purification methods, I examined triplicate metagenomes from a single ocean sample using four protocols. Concentration protocols showed statistical differences in taxonomy whereas purification protocols did not. Specifically, TFF-concentrated metagenomes contained trace bacterial contamination and had fewer abundant taxa as compared to FeCl₃-precipitated metagenomes. K-mer analysis using the complete dataset revealed polymerase choice defined access to "rare" sequences.To explore unknown viral sequences, I organized known and unknown sequence space into 27K high-confidence protein clusters (PCs) from 32 diverse Pacific Ocean Virus (POV) metagenomes, which doubled available PCs and included the first pelagic deep-sea viral metagenomes. Using PCs as a whole-viral-community diversity metric revealed decreases from coastal to open ocean, winter to summer, and deep to surface, that correlate with data from microbial genetic diversity markers (no parallel viral markers exist).Biologically, POV metagenomes showed that viruses likely reprogram central metabolic pathways in microbial communities far beyond the "photosynthesis viruses" paradigm. Gene distribution patterns from 35 viral gene families (31 new) revealed niche-specific (photic vs aphotic zone) altered pathway carbon flux presumably optimized to best locally generate energy and drive viral replication. Further, these PCs define the first "core" (180 genes) and "flexible" (423K genes total) viral community genome. Functionally, core genes again suggest niche-differentation with extensive Fe-S cluster-related genes for electron transport and metabolic enzyme catalysis in photic samples, and manipulation of host pressure-sensitive genes in aphotic samples. Taxonomically, these data deconstruct the culture-based paradigm that tailed viruses dominate in the wild - instead they appear ubiquitous, but not abundant.

The interaction between figs (Ficus spp.) and their pollinating wasps (fig wasps; Chalcidoidea, Hymenoptera) is a classic example of an ancient and apparently stable mutualism. A striking property of this mutualism is that fig wasps consistently oviposit in the inner flowers of the fig syconium (gall flowers, which develop into galls that house developing larvae), but typically do not use the outer ring of flowers (seed flowers, which are pollinated and develop into seeds). This dissertation explores the potential differences between gall and seed flowers that might influence oviposition choices, and the unknown mechanisms underlying gall formation. To identify the microbial community that could influence oviposition choice, I identified fungi in both flower types across six species of Ficus. I found that whereas fungal communities differed significantly as a function of developmental stages of syconia and lineages of fig trees, communities did not differ significantly between receptive gall and seed flowers. Because secretions from the poison sac that are deposited at oviposition are thought to be important in gall formation by both pollinating fig wasps and non-pollinating, parasitic wasps, I examined poison sac morphology in diverse galling wasps from several species of Ficus in lowland Panama. I found that the size of the poison sac was positively associated with egg number across pollinating and non-pollinating fig wasps. Finally to determine difference in defense and metabolism between gall and seed flowers, and to identify genes involved in galling, I compared gene expression profiles of fig flowers at the time of oviposition choice and early gall development. I found a prominence of flavonoids and defensive genes in both pollinated and receptive gall flowers of Ficus obtusifolia, and revealed detectable differences between gall flowers and seed flowers before oviposition. Several highly expressed genes were also identified that have implications for the mechanism of gall initiation. This dissertation explores previously unstudied aspects of the fig and fig wasp mutualism and provides important molecular tools for future study of this iconic and ecologically important association.

Recent studies suggest that horizontal gene transfer (HGT) plays an important role in niche adaptation in some eukaryotes and may be a major evolutionary force in unicellular lineages. One subcategory of HGT is endosymbiotic gene transfer (EGT), which is characterized by a large influx of genes from endosymbiont to host nuclear genome and is a critical step in the establishment of permanent organelles, such as plastids. The dinoflagellates are a diverse group of mostly marine eukaryotes that have a propensity for both HGT and plastid endosymbiosis. Many dinoflagellates are predators and can acquire both genes and plastids from prey, blurring the distinction between HGT and EGT. Here, I measure genome mosaicism in dinoflagellates to investigate how HGT has impacted gene innovation and plastid endosymbiosis in this group. Because analysis of HGT depends on accurate phylogenetic trees, I first assessed the sensitivity of automated phylogenomic methods to variation in taxon sampling due to homolog selection parameters. Using methods based on this analysis, I showed that a large amount of HGT has occurred in dinoflagellates, particularly from bacterial donors. Further, I demonstrated that the dinoflagellate Alexandrium tamarense has the largest number of genes gained relative to related eukaryotes using ancestral gene content reconstruction. Additionally, dinoflagellates have lost several ubiquitous eukaryotic metabolic genes, but missing genes have been functionally replaced by xenologs from many evolutionarysources. Other transferred genes are involved in diverse functions. These results suggest that dinoflagellate genomes are heavily impacted by HGT. Also, I investigated the timing and consequences of HGT in plastid endosymbiosis. Using the dinflagellate Dinophysis acuminata, a mixotrophic species that sequesters and maintains prey plastids, I identified plastid-targeted proteins that function in photosystem stabilization and metabolite transport. Dinophysis acuminate may be able to extend the useful life of the stolen plastid by protecting the photosystem and replacing damaged transporters. Phylogenetic analyses showed that genes are derived from multiple sources indicating a complex evolutionary history involving HGT. Dinophysis acuminate can acquire both genes and plastids from prey, which suggests that HGT could play an important role in plastid acquisition during the earliest stages of this transition.

Adaptive phenotypic plasticity is a key mechanism by which organisms deal with variation in many different aspects of their environment. Adaptive plasticity can occur in any trait, from aspects of biochemistry and morphology to behaviors. Because so many different traits can be plastic, organisms often respond plastically to a given change in their environment, such as an increase in temperature, with adaptive changes in multiple traits. Nevertheless, how these different plastic responses interact with each other and evolve together has received little attention. My research addresses these potential interactions among plastic traits and proposes new hypotheses regarding the causes and consequences of these interactions. It does so by focusing on heat avoidance in the caterpillars of Battus philenor (the pipevine swallowtail) which involves two distinct plastic mechanisms. First, the caterpillars can change color when they molt, a form of morphological plasticity in which they develop a red color under high temperatures which cools them by absorbing less solar radiation. Second, when the caterpillars become too hot, they will leave their host to seek cooler thermal refuges, a case of behavior as a form of plasticity. In terms of function, I demonstrated through field research that these two responses to high temperatures are largely redundant. Behavior provides a much stronger and faster response than color change, and red coloration provides little additional cooling when on a refuge. Instead, the primary benefit of color change is that it reduces the use of refuge seeking behavior, allowing the caterpillars to stay on their hosts longer. Using laboratory experiments, I demonstrated that this change in the use of refuge-seeking behavior with color occurs because color changes the cue for the behavior, body temperature, rather having any effect on how the caterpillar responds to that cue. Alternatively, similar experiments on caterpillars of varying sizes show that developmental size change lowers the body temperature at which caterpillars leave their host, demonstrating a change in the response to the cue (although larger caterpillars are also warmer, so both mechanisms are likely relevant for how size changes the expression of behavior). All of my research to this point was conducted on the local population in southern Arizona, which experiences quite high temperatures, but B. philenor is also found in much cooler environments, such as the Appalachian Mountains. Given this variation in their thermal environment, I used common garden experiments to compare the capacity for color change and refuge-seeking among B. philenor caterpillars from across the species range. Both color change and refuge seeking not only occurred in all populations, but also had the same reaction norms, occurring at the same temperatures and to the same degree. This is particularly notable for color change, which is not observed in the wild in northeastern populations, and thus has persisted despite minimal if any use. Overall, I have shown that studies of plasticity need to account for plasticity in different traits as well as the interactions between these forms of plasticity. My research on B. philenor provides a model for how to address these interactions, which future research can extend to additional organisms and environmental circumstances.

Social insects are used as models for understanding the evolution of sociality because they show seemingly complex behavioral and physiological traits that enforce group cohesion, collective organization, and group level reproduction. Social organization in insect societies requires workers to share information. Information sharing allows workers to efficiently perform and switch among tasks to meet colony needs. For many species that nest in preformed cavities, colony growth results in crowding inside the nest which can affect colony productivity and fitness. How does colony size and social density affect individual and collective behavior? Using a combination of laboratory and field experiments, I have begun to answer this question. In Temnothorax rugatulus ants, high social density resulted in greater colony energy use. In addition, larger colonies used proportionally less energy compared to smaller colonies, but showed reduced brood production. These results indicate that the way colonies use energy changes with social density and group size. In analyzing the effects of colony size and density on worker behavior, I found that high density increased worker connectivity and information sharing. Workers in larger colonies showed less connectivity compared to workers in smaller colonies. Interestingly, workers with more interactions spent less time in brood care. This study shows that workers' access to information and the overall pattern of information flow are affected by social density and colony size, and changes in worker connectivity can influence task behavior. The next study shows that field colonies maintained a relatively constant level of intranidal density irrespective of colony size; this suggests that Temnothorax ants actively regulate social density. When colonies were established in high density nests, they showed greater foraging and scouting activities, and this led to a higher probability for becoming polydomous, i.e., occupying multiple nests. When polydomy occurred, colonies divided evenly between two nests, but distributed fewer, heavier workers and brood to the supplemental nests. Taken together, the first four studies indicate that social density is an important colony phenotype that affects individual and collective behavior and energetics in ants, and the collective management of social density may be a group adaptation in ants and other social insects. Lastly, because crowding affects polydomy behavior, the final two experiments tested whether colony emigration and nest construction and dispersion, two strategies for reducing intranidal crowding, are influenced by food distribution. Temnothorax colonies preferred to emigrate to nests positioned closer to food, and weaver ants (Oecophylla smaragdina) positioned newly constructed nests in food-rich areas. Furthermore, weaver ants used the newly constructed nests to more rapidly retrieve and safeguard valuable food items. Thus, strategic emigrations and adaptive nest dispersion can remedy intranidal crowding and at the same time allow growing colonies to acquire adequate food to meet colony needs.

Between individual behavioral variation has been described in nearly every animal taxa where it has been measured. Often, these behavioral variations correlate across contexts, forming a behavioral syndrome. Despite a recent push to better understand the origins and consequences of behavioral syndromes, there still is no cohesive framework that describes this phenomenon. Here, I develop a social insect species into a model for measuring and testing behavioral syndromes at a new level of biological organization; the colony. This builds upon the rich literature describing between-colony variation in behavior and provides novel insights into the evolution of behavioral syndromes. In my first chapter I show that colonies do not vary from one another in foraging distance, nor is foraging distance directly associated with colony size. This was my first step in demonstrating that colony behavioral variation is not simply a byproduct of colony size. In chapter two, I expanded upon this finding by testing colonies both in the lab and in the field for a variety of ecologically relevant behaviors. Here, I found that there was a behavioral syndrome that reflected foraging distance, foraging effort to novel and familiar resources, response to threat and aggression. While there is a gradient of phenotypes, some colonies either travel farther to forage for food and respond more aggressively when confronted with a conspecific invader, but appear to invest less in each given incident or food source. I consider this to be more risk-tolerant; they increase their risk of external mortality for potentially larger pay-offs. On the other hand, risk-averse colonies deploy more foragers to exploit closer resources, increase their overall activity in the response to threat, but avoid travelling farther distances or aggressively engaging invaders. Additionally, there is between population variations in risk-taking phenotype. Across the western United States, colonies at more northern latitudes are more risk-tolerant than colonies at more southern latitudes. In chapter 3, I expand upon this latitudinal gradient in behavioral phenotype by investigating what ecological factors predict a colonies level of risk-tolerance. Specifically, I focused on ecological traits that reflected predation, competition, food resource availability and abiotic stress. I found that competition for nest sites and spatial clustering predicted behavioral type; colonies at high levels of nest site competition or spatial clustering were more risk-tolerant than colonies at lower levels of competition or were more spatially dispersed. In chapter 4, I used a common garden and brood transfer experiment to investigate if the relationship between the ecological environment and behavior was the result of phenotypic plasticity or local adaptation. I show that local adaptation is the most likely explanation, as colonies with more workers from the donor colony are more, behaviorally, like the donor colony than colonies with fewer donor workers. In chapter 5 I test if the risk-taking behavioral syndrome is the result of life history strategy variation. I test the growth rate and energy allocation towards either somatic effort or reproductive effort. I found that colonies which are risk-tolerant also grow faster and dedicate more energy towards reproductive effort, which is consistent with predictions built from life history theory. This body of work shows that behavioral syndromes can exist at a new level of organization, the colony, and that variation in behavioral type is the result of differential selection pressure between populations. This directly connects behavioral syndrome research to life history strategy research. As life history strategy theory is a well-understood field, this represents a true advancement in the field of behavioral syndromes.