The communities of microorganisms associated with humans and other animals are characterized by a large degree of diversity and unexplained variation across individual hosts. While efforts to explain this variation in host-associated systems have focused heavily on the effects of host selection, community assembly theory emphasizes the role of dispersal and stochastic demographic processes, otherwise known as ecological drift. In this dissertation, I characterize the communities of microorganisms associated with the zebrafish, Danio rerio, intestine, and assess the importance of microbial dispersal and drift to their assembly. First, I describe changes in the composition and diversity of the zebrafish intestinal microbiome over zebrafish development and show that while host development is a major driver of community composition over time, there remains a large amount of unexplained variation among similar hosts of the same age. I go on to show that random dispersal and ecological drift alone in the absence of host selection are sufficient to explain a substantial amount of this variation, but the ability of these processes to predict the distribution of microorganisms across hosts decreases over host development. Finally, I present an experimental test of dispersal in host-associated systems, and show that not only does dispersal among individual zebrafish hosts have a large impact on the composition and diversity of associated microbial communities, but it can also overwhelm the effects of important host factors, such as the innate immune system. As a whole, this work demonstrates that the composition and diversity of microbial communities associated with animal hosts are not solely the result of selection by the host environment, but rather dispersal and stochastic processes have important and often overwhelming effects on their assembly. To fully understand the assembly of host-microbe systems, we must broaden our focus to include scales beyond that of an individual host and their associated microorganisms. / 10000-01-01

Doctor of Philosophy / Genetics Interdepartmental Program - Biology / Susan J. Brown / Understanding evolutionary processes that structure genetic variation associated with lineage diversification and speciation is a central problem. Shifts in host plant use by insect herbivores and subsequent divergence in mating signals can lead to sexual isolation, especially when selection for specialization on different hosts restricts gene flow among populations. The grasshopper Hesperotettix viridis (Thomas) is an oligophagous grasshopper feeding on plants primarily in the host plant genera Gutierrezia and Solidago in Kansas. I used mitochondrial and microsatellite genetic markers to evaluate the diversification pattern and underlying evolutionary mechanisms of two putative host races of H. viridis. I also quantified host preferences, the degree of sexual isolation among putative host races and divergence in cuticular attributes to identify the nature and origins of initial barriers that isolated populations in the formative stages of divergence. mtDNA data revealed a star-shaped phylogeny, suggesting isolation in a single refugium ~110,000 years ago based on a molecular clock, followed by rapid population expansion. Microsatellite data reveal significant host-based genetic differentiation and structuring in H. viridis populations in Kansas, including a microsatellite locus under strong divergent selection. Neutral microsatellite loci did not reveal a differentiation pattern specific to host plant use. Significant host-based preferences by individuals that fed on two host plant groups were detected in host paired-feeding preference studies. No-choice mate selection experiments revealed preferences for individuals collected from the same host species independent of location with little mating observed between individuals from different host species. Significant differentiation in color and cuticular composition among different host plant races within the study area was also detected. Correlations between host choice, mate choice and phenotypic divergence were observed and this host associated divergence appears to have a genetic basis. Based on the results of this study, I conclude that divergent selection for host plant use underlies observed sexual isolation among populations in this species. Hesperotettix viridis populations in Kansas that fed on Solidago and Gutierrezia species represent two incipient host races, early stages of diversification that could lead to speciation in insect herbivores.

Prior exposure to plants cues can enhance assortative mating in insects. We hypothesized that, as previously reported for females, males of Cotesia congregata would display inherent responses to plant cues that could be modified by postemergence experience and further, that males originating from two different host-plant complexes (HPCs) would display different behavioral responses to these HPCs. In no-choice contact assays with a non-host plant, searching responses of males and females increased sharply at Day 2 and remained stable through Day 4. In no-choice assays with potential host plants, males searched longer on catalpa than tobacco; responses were not modified by postemergence experience. In choice assays with both HPCs, naïve males did not display orientation preferences; however, males experienced with their natal plant preferred their natal HPC. Results indicate that postemergence experience on the natal host plant induces an orientation preference for the natal HPC and thus, can facilitate assortative mating.

The cotton fleahopper, Pseudatomoscelis seriatus (Reuter) is a widely distributed insect across the United States. Although, it feeds on several native wild hosts, its agricultural importance lies as an economic pest of cotton in several states in the southern United States. No studies have addressed intraspecific genetic and phenotypic variation of this insect pest at a large geographic scale. I examined genetic variation among cotton fleahopper populations associated with cotton in different geographic locations across the southern United States (Chapter II). Using dominant, neutral, nuclear molecular markers (AFLP, amplified fragment length polymorphism) and mitochondrial DNA sequences, I found that overall genetic differentiation among different geographic populations, collected from cotton in eleven cotton growing states, was low but significant. AFLP revealed the presence of three regional groups representing western (Arizona), central (Texas, Oklahoma, Arkansas, Louisiana, Mississippi and Alabama), and eastern (Florida, Georgia, South Carolina and North Carolina) populations. I examined if there were distinct lineages of cotton fleahoppers associated with three of its host plant species: cotton (Gossypium hirsutum), horsemint (Monarda punctata) and woolly croton (Croton capitatus) in five different locations of Texas by using AFLP markers (Chapter III). I found two distinct host-associated lineages at three locations and local panmixia in the other two locations. I tested if host preference of cotton fleahoppers were affected by geographic variation and prior experience. Conducting choice tests with a Y-tube olfactometer, I found that host preference in cotton fleahoppers for horsemint (one of its native host plants) is conserved and unaffected by individual?s prior experience with cotton (Chapter IV). Finally, I explored the role of host-plant species in morphological differentiation of the cotton fleahopper in two locations that differ in presence of distinct host-associated lineages. Using a geometric-morphometric approach, I detected significant effect of host plant and geography on body morphology and wing shape of cotton fleahopper populations (Chapter V). Length of antenna and rostrum were two important traits associated with morphological divergence of cotton and horsemint associated insect populations. Cotton associated individuals had relatively longer antenna and rostrum compared to individuals associated with horsemint.

Emergent infectious diseases are a major driver of the accelerated rates of biodiversity loss that are being documented around the world. Global losses of amphibians provide evidence of this, especially those associated with chytridiomycosis, a lethal skin disease caused by the fungus Batrachochytrium dendrobatidis (Bd). Amphibian skin can harbor diverse bacterial communities that, in some cases, can inhibit the growth of Bd. Thus, there is interest in using skin bacteria as probiotics to mitigate Bd infections in amphibians. However, experiments testing this conservation approach have yielded mixed results, suggesting a lack of understanding about the ecology of these microbial communities. My dissertation research aimed to assess basic ecological questions in microbial ecology and to contribute to the development of probiotics using amphibian skin bacteria. First, to assess whether environmental conditions influence the function of amphibian skin bacterial communities, I conducted a field survey across low and high elevation populations of an amphibian host to assess their skin bacterial communities and metabolite profiles. I found that similar bacterial communities produced different metabolites at different locations, implying a potential functional plasticity. Second, since culturing is critical for characterizing bacteria, I aimed to identify the culture media (low vs high nutrient concentration) that recovers the most representative fraction of the amphibian skin bacterial community. I found that media with low nutrient concentrations cultured a higher diversity and recovered a more representative fraction of the diversity occurring on amphibian skin. I also determined that sampling more individuals is critical to maximize culture collections. Third, I assessed the diversity of the amphibian skin fungal community in relation to Bd infection across eight amphibian species. I determined that amphibian species was the most important predictor of fungal diversity and community structure, and that Bd infection did not have a strong impact. My dissertation highlights the importance of environmental conditions in the function of amphibian skin bacteria, expands our knowledge of the understudied fungal component of the amphibian skin microbiome, and complements current efforts in amphibian conservation. / Ph. D. / In light of the global losses of amphibian diversity due to, in part, the skin disease chytridiomycosis (caused by the fungus Batrachochytrium dendrobatidis [Bd]); the discovery that some amphibian-skin bacteria can inhibit Bd growth provides hope for amphibian conservation via their use as probiotics to control Bd infections. However, experiments testing these bacteria have yielded inconsistent results, suggesting a limited understanding about the factors influencing the diversity of amphibian-skin microbes and their ability to inhibit Bd. Also, efforts to identify effective candidates for probiotic therapy are still premature. Thus, my dissertation had an ecological emphasis and focused on complementing conservation efforts focused on probiotics. First, I assessed whether environmental conditions influence bacteriallyproduced products, which can have antifungal properties. Specifically, I surveyed low and highelevation populations of an amphibian species to assess the skin-bacteria and their products. I determined that, while skin bacterial communities were similar across an environmental gradient, their products differed, suggesting potential different antifungal properties. Second, I assessed the ability of different culture media types (low vs high nutrient concentrations) to grow a high portion and most representative fraction of the amphibian-skin bacteria. I found that culture media with low nutrient concentrations allowed the growth of a higher diversity of the bacteria occurring on the amphibian-skin, including the abundant members, and also determined that including a large number of amphibians is the best way to improve culture collections. Third, I assessed the fungal diversity occurring in the skin of different amphibian species and how it might response to Bd infections, and examined whether skin-fungi interact with co-occurring bacteria. I found that the amphibian species was the most important driver of the fungal diversity, and that Bd infection did not influence the diversity of these communities. Moreover, I identified the most diverse fungal phyla occurring in the amphibian-skin and determined that these fungi might interact with co-occurring bacteria. My dissertation contributes to our understanding about the influence of the environmental conditions in the amphibian-skin bacteria, expands our limited knowledge on the amphibian-skin fungi, and complement current amphibian conservation efforts.

Endoparasitoids of herbivorous lepidopterans have intimate relationships with their host species as well as the plant on which their host develops. Characteristics of both hosts and plants can affect parasitoid success in tri-trophic systems and thus, drive diversification. Genetic differentiation was estimated for Cotesia congregata (Say) collected from two distinct host-plant complexes, Manduca sexta L. on tobacco (Nicotiana tabacum L.) and Ceratomia catalpae, (Haworth) on catalpa (Catalpa bignonioides Walker), in the mid-Atlantic region of North America using seven microsatellite loci and the mitochondrial COI locus. Microsatellite allele frequencies were differentiated based on host-plant complex, and COI haplotypes from individuals on the same host-plant were identical despite geographic distances between catalpa sites of up to 830 km and distances between tobacco sites of up to 294 km. Results indicate genetic differentiation of subpopulations of C. congregata based on host-plant complex and not geographic distance, and were designated as host races. Cotesia congregata is a gregarious parasitoid, meaning that many individuals develop in a single host larva. Superparasitism, or repeated egg-laying events in the same host larva, is likely to occur in gregarious species. Brood size was not a good predictor of superparasitism in C. congregata, but within-brood male allele diversity indicates either superparasitism or multiple mating by female wasps.

Phytophagous insects make up over one quarter of described species on Earth, and this incredible diversity seems directly linked to feeding on plants. Comparative studies of sister groups have shown shifts to herbivory are consistently associated with increased species diversity in insects, but the reasons for this diversification remain unclear. While other explanations, such as decreased extinction rates or influences on population structure, exist, one prominent hypothesis suggests shifts and subsequent adaptation to novel host plants can lead to the evolution of reproductive barriers. Given their extreme specialization on host plants in the genus Pinus and intimate, life-long association with their host plants, divergent host use has been suspected to drive speciation in the conifer sawfly genus Neodiprion. Previous work showed host shifts coincide with speciation events in the genus; but could not determine if these host shifts initiated speciation or if they occurred after other reproductive barriers arose. Determining the contribution and timing of host shifts relative to speciation will require examination of populations at the earliest stages of divergence, before post-speciation changes amass. If host shifts frequently drive speciation in the genus, there will likely be evidence of host-driven divergence within species occurring on a wide range of host plants. The goal of this dissertation is to examine populations of the red-headed pine sawfly, Neodiprion lecontei, an abundant, well-studied pest species that occurs on multiple hosts throughout its range, for evidence of host-driven divergence. Using a combination of reduced representation genomic sequencing, population genomics, and ecological assays, I specifically look for evidence of 1) genetic differentiation between populations utilizing different host plants, 2) ecological divergence in female oviposition preference, larval performance, and ovipositor morphology between populations on different hosts, and 3) ecologically-driven reproductive isolation between genetically and ecologically divergent populations. Each chapter of this dissertation examines the role of host use in driving ecological, genetic, and/or reproductive divergence within N. lecontei at a different spatial scale. First, I surveyed range-wide patterns of diversity. I identified three genetic clusters, dated the divergence of these clusters to the late Pleistocene, and found evidence that both dispersal limitation (geography) and host use contribute to genetic differentiation within N. lecontei. Next, I looked within one of these genetic clusters for additional evidence of the role of host in driving divergence. Sawflies in this cluster primarily utilize two hosts which differ significantly in needle architecture. Although I found no evidence of neutral genetic differentiation between hosts exists, I did detect spatial and temporal differences in host use, and host-specific differences in ovipositor morphology, a performance-related trait. Finally, I examine a single site where N. lecontei utilizes three structurally divergent species of pine. Although there was little genetic structure, no sexual isolation, and no distinct host preferences, the host types were partially temporally isolated and varied in ovipositor morphology and larval performance across on the three hosts. Overall, although divergent host use consistently resulted in divergent ovipositor morphology, a reduction in gene flow via temporal or geographic isolation may be required before additional forms of ecological and genetic differentiation can develop. Together these results suggest host shifts alone may not be enough to drive population divergence and speciation in Neodiprion.

Chaque être vivant interagit avec une ou plusieurs espèces et est membre d'un réseau complexe d'interactions qui influencent les traits des individus, exerçant de fait des pressions sélectives sur leurs populations. Chaque espèce étant dépendante de la nature et de la diversité des interactions dans lesquelles elle est impliquée, son évolution est donc en partie liée aux autres espèces avec lesquelles elle interagit. Un hôte et son parasitoïde vivent dans une dynamique coévolutive, prenant part à une véritable 'course aux armements', où les différentes stratégies d'attaque peuvent être sélectionnés chez le parasitoïde en réponse aux différentes formes de défenses chez l'hôte. Ce dernier interagit également avec d'autres organismes qui modifient ses traits, impactant les aptitudes du parasitoïde, perturbant leur dynamique coévolutive. L'objectif de ce travail est ainsi d'identifier l'influence du réseau d'interactions du puceron du pois Acyrthosiphon pisum sur l'évolution et l'écologie des populations de son principal parasitoïde Aphidius ervi. Le puceron du pois est lui-même parasite de sa plante hôte, leur coévolution aboutissant à une spécialisation de cet aphide cette espèce hôte végétale. Les populations de pucerons sont donc structurées en races d'hôtes sympatriques, divergeant génétiquement et phénotypiquement. Notre étude montre une absence de structuration génétique des populations de parasitoïdes selon les races d'hôte. A. ervi exploite indifféremment les A. pisum issus de différentes plantes hôtes, excluant la présence d'un effet cascade associé à la spécialisation alimentaire chez ce puceron. La dispersion et le caractère généraliste du parasitoïde semblent favoriser les flux de gènes entre les différents éléments d'un paysage agricole. A. pisum peut également abriter le symbiote Hamiltonella defensa qui lui confère une résistance à A. ervi. Ces microorganismes symbiotiques induisent une réduction des défenses comportementales chez les pucerons porteurs. Cette diminution de l'expression des comportements défensifs favorise à la fois le puceron et son symbiote car elle réduit les coûts associés à ces comportements. Enfin, une évolution expérimentale sur des populations de parasitoïdes soumises à des populations d'hôtes portant H. defensa (résistantes) ou non (sensibles) montre une adaptation des parasitoïdes soumis aux hôtes résistants. Cette adaptation s'accompagne d'une réduction de la variabilité génétique dans les populations exposées à la résistance. On observe également une divergence génétique entre les populations exposées ou non à la résistance conférée par le symbiote, H. defensa. Cette expérimentation met en évidence le potentiel évolutif des populations d'A. ervi et donc leurs capacités d'adaptation face à des pucerons résistants. Les intérêts fondamentaux et appliqués de ces travaux sont discutés et replacés dans un contexte général.