The restoration of degraded tallgrass prairies can mitigate climate change due to the carbon accrued during the development of grasslands. The focal species, dominant grass Andropogon gerardii, can assist the recovery of grassland ecosystem functioning. Climate, local adaptation, and biodiversity have been found to impact the accrual of carbon in grasslands. This study examined the difference in ecosystem functioning between ecotypes along a dry to mesic precipitation scale. The study site for this project was at the Southern Illinois University Agriculture Research Center in Carbondale, Illinois. The field site was planted with seeds originating from dry to mesic ecotypes, and the resulting ecosystem functioning was analyzed. It was found that the Kansas non-local ecotypes had significantly higher biodiversity, while the local Illinois sites demonstrated local adaptation with A. gerardii. Aboveground plant biomass was higher in the local sites, but there was no difference in carbon accrual between any of the ecotypes. While ecotypic variation in a dominant species will usually differentially influence ecosystem functioning, in this case, high biodiversity and local adaptation result in similar carbon inputs in grassland soil. It is necessary to analyze the carbon content of the soil in the drier field sites in order to determine if major differences in rainfall leads to differences in carbon accrual. If the goal of restoring a tallgrass prairie in southern Illinois is to assist with climate change mitigation, then it does not make a significant difference if the dominant species is sourced locally or non-locally.

Wide-scale conversion of tallgrass prairie to row-crop agriculture has spurred restoration of this endangered ecosystem. At the onset of restoration, a matrix of native plant species is sown into former crop field and includes warm-season (C4) grasses, cool-season (C3) grasses, legumes, and a large variety of herbaceous forbs. Increased demand for native seed due to a greater number of areas targeted for restoration has increased use of C4 grass cultivars by restoration practitioners. Cultivars are selectively bred to display traits such as increased productivity and digestibility, thus highlighting their original use in rangelands of the Great Plains. C4 grasses have a mutualistic relationship with arbuscular mycorrhizal fungi (AMF). In remnant tallgrass prairie, AMF can increase C4 plant uptake of belowground resources (e.g., water, soil P) by increasing root surface area. It is unknown if AMF colonization varies between seed source (cultivar or non-cultivar) of C4 grasses used in restoration and if this further affects plant biomass. Intraspecific variation in AMF colonization between two dominant warm-season prairie grasses was tested in two established prairie restoration experiments, both having plots seeded with either C4 cultivars or non-cultivars. To test for effects of seed source and AMF colonization on plant biomass, a greenhouse experiment was designed using two source populations (cultivar and non-cultivar) of two species (Andropogon gerardii Vitman and Sorghastrum nutans (L.) Nash) and soil collected at each field restoration (Kansas and Illinois). To suppress activity and colonization of AMF, a fungicide (Allban Flo: Thiophanate Methyl) was applied to half of the containers. Warm-season (C4) grass cultivars had greater or equivalent biomass production than non-cultivars at the onset of field restoration and also in the greenhouse. Furthermore, cultivars generally had less or equivalent root colonization by AMF and dependence on fungicide-free soil was greater in cultivars to retain increased accrual of biomass. It was, however, not possible to determine the role of AMF in plant biomass production as fungicide did not successfully reduce AMF root colonization in cultivars or non-cultivars, with one exception. It is critical that an effective AMF-suppression treatment be established in these types of studies. Future experiments should validate supposed effectiveness of the newly-recommended fungicide (Topsin-M) in population sources of warm-season prairie grasses and also apply it to the soil at time of planting in greenhouse studies. In the field sites, adjacent soil cultivation may have contributed to greater AMF biomass more so than surrounding remnant prairie. Future research identifying species composition of AMF at these sites is necessary to clarify differences in biomass. Despite greater plant biomass in cultivars, soil nutrient availability remained equivalent between source populations in general. Available N and P were not less in soils grown with cultivars, however soil inorganic N was inversely related to root length colonized by AMF, suggesting a role of AMF in N transfer from soil to plant. Soil P was not different between source populations likely due to legacy effects of agricultural fertilization, thus limiting a well known benefit of AMF symbiosis, at least at the onset of restoration. Non-target effects of fungicide application were observed (e.g., changes in available N) and effectiveness of AMF suppression was questionable. Fungicide lowered pH and increased N availability in soil as indicated by main effects of application and a positive relationship between pH and inorganic N across species. Fungicide application either 1) decreased N uptake by soil microorganisms (possibly including AMF) or 2) increased competition for adsorption sites and/or solubility of total inorganic N as pH changed, thus making this nutrient more available in the soil solution. Future examination quantifying indirect effects of fungicide application on soil chemistry should also be considered to better elucidate role of AMF in plant growth and soil nutrient availability between cultivars and non-cultivars of warm-season grasses used in tallgrass prairie restoration.

Master of Science / Statistics / Nora M. Bello / Big bluestem (Andropogon gerardii) is a wide-ranging dominant prairie grass of ecological and agricultural importance to the US Midwest while edaphic subspecies sand bluestem (A. gerardii ssp. Hallii) grows exclusively on sand dunes. Sand bluestem exhibits phenotypic divergence related to epicuticular properties and enhanced drought tolerance relative to big bluestem. Understanding the mechanisms underlying differential drought tolerance is relevant in the face of climate change. For bluestem subspecies, presence or absence of these phenotypes may be associated with RNA transcripts characterized by low number of read counts. So called low-count transcripts pose particular inferential challenges and are thus usually filtered out at early steps of data management protocols and ignored for analyses. In this study, we use a plasmode-based approach to assess the relative performance of alternative inferential strategies on RNA-seq transcripts, with special emphasis on low-count transcripts as motivated by differential bluestem phenotypes. Our dataset consists of RNA-seq read counts for 25,582 transcripts (60% of which are classified as low-count) collected from leaf tissue of 4 individual plants of big bluestem and 4 of sand bluestem. We also compare alternative ad-hoc data filtering techniques commonly used in RNA-seq pipelines and assess the performance of recently developed statistical methods for differential expression (DE) analysis, namely DESeq2 and edgeR robust. These methods attempt to overcome the inherently noisy behavior of low-count transcripts by either shrinkage or differential weighting of observations, respectively. Our results indicate that proper specification of DE methods can remove the need for ad- hoc data filtering at arbitrary expression threshold, thus allowing for inference on low-count transcripts. Practical recommendations for inference are provided when low-count RNA-seq transcripts are of interest, as is the case in the comparison of subspecies of bluestem grasses. Insights from this study may also be relevant to other applications also focused on transcripts of low expression levels.

Ecological theory predicts that high environmental heterogeneity causes high biodiversity. Theory further predicts that more biodiversity results in greater ecosystem functioning. These theoretical predictions were evaluated in three studies using grassland restorations from agriculture.

Root productivity likely has consequences for the composition, activity, and recovery of soil microbial populations and the belowground processes mediated by these organisms. In tallgrass prairie, ecotypic variation potentially exists in response to a strong precipitation gradient across the Great Plains. Thus, ecotypic variation within a species may differentially affect belowground net primary productivity (BNPP), the associated soil microbial community, and may scale up to affect ecosystem processes. The goals of this study were to elucidate: (1) whether ecotype, environment, or an ecotype by environment interaction regulate BNPP of a dominant species (Andropogon gerardii) collected from and reciprocally planted in common gardens across a precipitation gradient, and (2) whether variation in BNPP scales to affect microbial biomass and ecosystem processes. I quantified root biomass, BNPP (using root ingrowth bags), soil microbial biomass, and nutrient mineralization rates in root-ingrowth cores below six population sources of A. gerardii (2 Illinois, 2 eastern Kansas, and 2 central Kansas) established in southern Illinois, eastern Kansas, and central Kansas. An ecotype effect was found on above and belowground net primary productivity, but these findings did not translate to soil response variables. Microbial populations themselves may affect the productivity and composition of prairie species. In a second study, soil ecological knowledge (SEK) was tested by applying a native prairie soil slurry amendment to restoration plots to determine efficacy of this method as a restoration practice. The goals of this two year study were to elucidate: (1) whether a slurry amendment of prairie soil would increase above and belowground productivity and belowground ecosystem processes in a prairie restoration, and (2) to evaluate whether differences in plant diversity will scale to affect belowground productivity and ecosystem processes. I quantified aboveground net primary productivity (ANPP) and species composition, as well as root biomass, belowground net primary productivity (BNPP), soil microbial biomass, and nutrient mineralization rates in root-ingrowth cores installed in treated and control plots. A treatment effect was noted on root biomass and total PLFA biomass; however, there was no treatment effect on cover, ANPP, or soil microbial processes. Though the soil microbial community did represent native prairie soil, there was poor establishment of prairie plant species. These factors may be due to the limited time available for data collection and the lack of precipitation in the second growing season. Longer studies may be necessary to fully examine the effects of soil slurry amendments as restoration tools.

The tallgrass prairie has been severely reduced in size, making restoration important to maintain communities and functions of this ecosystem. A chronosequence approach was used to determine recovery of physical and biological soil properties. The recovery models of soil properties provided information to explain the variation in total C stock of the whole soil. Recovery models also provided information to design a competition experiment based on variation in whole soil conditions with land use history. The filter framework hypothesis is a useful concept for examining tallgrass prairie restoration; the theory states only a subset of species in the region will be able to establish in a specific location due to abiotic and biotic filters. With this theory in mind, I explored the influence of whole soil conditions as affected by land use history (cultivation/restoration) and how these conditions altered plant-plant competition dynamics of a dominant grass was studied. Belowground plant biomass recovers with cessation of tillage and restoration back to prairie, providing an organic matter source for microbial populations to recover and soil macroaggregates to form. This has potential to increase C sequestration in soils and decrease nitrous oxide efflux from soils. Intact 5.5 cm dia cores were collected to a depth of 10 cm in each field to determine physical and biological soil properties. Belowground plant, microbial community, and soil structure properties were modeled to recover coinciding with an increase in total C stock of the whole soil. Structural equation modeling revealed that soil structure physically protecting organic matter explained the most variation in soil carbon sequestration with restoration. Most of the total C was contained within the macroaggregate size fraction; within this fraction most of that C is within the microaggregates within macroaggregates fraction. Soil structure is critical for recovery of soil carbon stocks and the microaggregate within macroaggregate fraction is the best diagnostic of sequestered C. ANCOVA results indicate that while the slopes of nitrous oxide efflux rates did not differ, cumulative efflux differed, though this was not related to time since restoration. Dominant grasses, such as Andropogon gerardii, can exclude subordinate species from grassland restorations. Thus, understanding changes in competition dynamics of dominant grasses could help maintain richness in grassland restorations. There may be changes in competition dynamics with whole soil conditions affected by land use history (cultivation/restoration) as plant available nutrients will decrease, microbial populations will increase, and soil structure will improve with restoration from cultivation to prairie. Using 4 soil treatments of varying land use history with four species treatments, to determine if effects are general or species specific, pairwise substitution competition experiments were conducted. Relative A. gerardii response to competition was compared among soil and species treatments using competition intensity and competition importance indices utilizing final plant biomass, relative growth rate based on maximum height, and net absolute tiller appearance rate. The experiment was conducted over 18 weeks, allowing A. gerardii to flower. A significant intensity result and significant importance results utilizing biomass measurements indicated that the 16 year restored prairie soil cause A. gerardii to be a relatively better competitor against forbs than in all other soils except for cultivated soil, likely due to positive plant-soil feedbacks. Significant importance results utilizing tiller appearance rate indicated that the cultivated and 3 year restored prairie soil caused A. gerardii to be a relatively better competitor than in the 16 year restored and never cultivated native prairie soils, likely due to changes in whole soil conditions related to land use history. There were only general soil effects, as soil treatments did not interact with species treatments. A. gerardii was a relatively better competitor against non-leguminous forbs, indicating that legumes are a better competitor for a limiting nutrient than A. gerardii or that this species is not in direct competition with legumes.

Master of Science / Department of Plant Pathology / Karen A. Garrett / While plants are typically exposed to multiple stressors in the field, studies of genome-wide gene expression and phytohormone responses in wild plant species exposed to multiple stressors are rare. Our objectives were to determine the effects of drought and rust stress on gene expression in Andropogon gerardii, the dominant grass in tallgrass prairie, and associated levels of phytohormone production. In a factorial design, plants experiencing drought or non-drought conditions were either inoculated with the rust pathogen Puccinia andropogonis or not inoculated. Gene expression was evaluated with maize microarrays. Drought-stressed plants significantly decreased expression of genes associated with photosynthesis and the hypersensitive response, while expression of genes associated with chaperones and heat-shock proteins increased. No significant differences in gene expression in response to the rust treatment were detected using a mixed model analysis of variance and false discovery rate protection, probably because of the low infection rate. Phytohormone production increased when both stresses were present. The rust treatment significantly increased benzoic acid (BA) production in the presence of drought, while the drought treatment alone significantly increased salicylic acid (SA) production. Leaf tips usually had higher levels of all phytohormones in all treatments and the leaf section evaluated had a larger effect on phytohormone level than did the treatments applied.

Master of Science / Department of Plant Pathology / Karen A. Garrett / Ecological clines often result in gradients of disease pressure in natural plant communities, imposing a gradient of selection on disease resistance genes. We describe the diversity of a resistance gene homolog in natural populations of the dominant tallgrass prairie grass, Andropogon gerardii, across a precipitation gradient ranging from 47.63 cm/year in western Kansas to 104.7 cm/year in central Missouri. Since moisture facilitates infection by foliar bacterial pathogens, plants along this precipitation gradient will tend to experience heavier bacterial disease pressure to the east. In maize, the gene Rxo1 confers resistance to the pathogenic bacterium Burkholderia andropogonis. Rxo1 homologs have been identified in A. gerardii and B. andropogonis is known to infect natural populations of A. gerardii. The spatial genetic structure of A. gerardii was assessed from central Missouri to western Kansas by genotyping with AFLP markers. Samples were also genotyped for Rxo1 homologs by amplifying an 810 base pair region of the leucine-rich repeat and digesting with restriction enzymes. We compared Rxo1 homolog diversity to AFLP diversity across different spatial scales. Genetic dissimilarity based on AFLP markers was lower than would have occurred by chance at distances up to 30 m, and different prairies were more dissimilar than would have occurred by chance, but there was not a longitudinal trend in within-prairie dissimilarity as measured by AFLP markers. Dissimilarity of the Rxo1 homologs was higher in the east suggesting the presence of diversifying selection in the more disease-conducive eastern environments.