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The structure and reactivity of electrosprayed protein ions is governed by their net charge. Native proteins in non-denaturing aqueous solutions produce low charge states. More highly charged ions are formed when electrospraying proteins that are unfolded and/or exposed to organic supercharging agents. Numerous studies have explored the electrospray process under these various conditions. One phenomenon that has received surprisingly little attention is the charge enhancement caused by multivalent metal ions such as La3+ when electrospraying proteins out of non-denaturing solutions. Here, we conducted mass spectrometry and ion mobility spectrometry experiments, in combination with molecular dynamics (MD) simulations, to uncover the mechanistic basis of this charge enhancement. MD simulations of aqueous ESI droplets reproduced the experimental observation that La3+ boosts protein charge states relative to monovalent metals (e.g., Na+). The simulations showed that gaseous proteins were released by solvent evaporation to dryness, consistent with the charged residue model. Metal ion ejection kept the shrinking droplets close to the Rayleigh limit until ∼99% of the solvent had left. For droplets charged with Na+, metal adduction during the final stage of solvent evaporation produced low protein charge states. Droplets containing La3+ showed a very different behavior. The trivalent nature of La3+ favored adduction to the protein at a very early stage, when most of the solvent had not evaporated yet. This irreversible binding via multidentate contacts suppressed La3+ ejection from the vanishing droplets, such that the resulting gaseous proteins carried significantly more charge. Our results illustrate that MD simulations are suitable for uncovering intricate aspects of electrospray mechanisms, paving the way toward an atomistic understanding of mass spectrometry based analytical workflows.

It is a challenge to both use and conserve wild-harvested medicinal plants, especially when it appears they may be threatened by harvest pressure, and there is often limited biological information available to inform management decisions. Osha (Ligusticum porteri) is an important medicinal plant whose roots are harvested in the southwest USA and Mexico as an herbal remedy to treat flu, sore throat, and other illnesses. We studied population structure, root production, and the ability of osha to recover from harvest in different environmental contexts at two high-elevation southern Colorado sites with a goal of understanding what a sustainable rate of harvest might be. We experimentally harvested roots of mature osha plants at four different rates. Results indicate that low rates of harvest allow for stable osha populations over the short term (3–5 year) at our sites. Due to management interest by the USDA Forest Service, we propose a possible sustainable harvest rate of 50% of mature plants every 10 years. Given variability due to weather and other environmental factors, we recommend that future osha harvest should be planned and adjusted after careful monitoring.

It is well-known that supercharging agents (SCAs) such as sulfolane enhance the electrospray ionization (ESI) charge states of proteins, although the mechanistic origins of this effect remain contentious. Only very few studies have explored SCA effects on analytes other than proteins or peptides. This work examines how sulfolane affects electrosprayed NaI salt clusters. Such alkali metal halide clusters have played a key role for earlier ESI mechanistic studies, making them interesting targets for supercharging investigations. ESI of aqueous NaI solutions predominantly generated singly charged [NanI(n-1)]+ clusters. The addition of sulfolane resulted in abundant doubly charged [NanI(n-2)Sulfolanes]2+ species. These experimental data for the first time demonstrate that electrosprayed salt clusters can undergo supercharging. Molecular dynamics (MD) simulations of aqueous ESI nanodroplets containing Na+/I- with and without sulfolane were conducted to obtain atomistic insights into the supercharging mechanism. The simulations produced [NanIi]z+ and [NanIiSulfolanes]z+ clusters similar to those observed experimentally. The MD trajectories demonstrated that these clusters were released into the gas phase upon droplet evaporation to dryness, in line with the charged residue model. Sulfolane was found to evaporate much more slowly than water. This slow evaporation, in conjunction with the large dipole moment of sulfolane, resulted in electrostatic stabilization of the shrinking ESI droplets and the final clusters. Hence, charge-dipole stabilization causes the sulfolane-containing droplets and clusters to retain more charge, thereby providing the mechanistic foundation of salt cluster supercharging.

Traditional foods of indigenous people are a potential untapped source for providing nutritious dietary options in the modern world. The rising popularity of indigenous foods such as amaranth in recent years suggests that these foods can become a popular part of a healthy diet in the United States. Traditional knowledge of indigenous people can form the basis for using wild food resources. This research provides data on 50 plant species common to the central United States that are abundant sources of dietary protein and fiber, and used traditionally by Native American tribes. Many species show promise with high amounts of protein and fiber being found in lamb’s quarters, nettles, grass seeds, and buffalo gourd seeds. These results honor traditional knowledge. In addition, many of these species could be cultivated using techniques similar to those existing for commercial grains and vegetables. Seeds and/or greens of these wild plants could easily be incorporated into the diets of many more people, improving modern diets, and the nutritional quality of food products.

1. Novel ecosystems can differ from the native systems they replaced. We used phenology measures to compare ecosystem functioning between novel exotic-dominated and native-dominated grasslands in the central US. 2. Phenology, or timing of biological events, is affected by climate and land use changes. We assessed how phenology shifts are being altered by exotic species dominance by comparing remotely sensed Normalized Difference Vegetation Index (NDVI) within growing seasons at exotic- and native-dominated sites along a latitudinal gradient. Exotic species were dominated by the C3 species functional group in the north and the C4 species functional group in the south. 3. Date of senescence was an average of 36 days later in exotic than native-dominated grasslands, and this effect was consistent across latitudes. 4. Exotic-dominated grasslands greened up an average of 10.7 days earlier than native-dominated grasslands, but this effect was highly dependent on latitude and the plant functional group that dominated at that latitude. Greenup differed between native and exotic sites the most in central and northern regions that had dominant C3 grasses. 5. We estimated the effects of an increase in global temperatures on green-up and senescence with a space-for-time substitution, and by comparing growing degree day differences between historical average temperatures and +2.5° C. Green-up was significantly earlier and senescence was significantly later with a 2.5 ° C increase in temperature. The native-exotic difference was significantly greater than the difference due to increased temperature for senescence, but not for green-up. 6. Synthesis and applications. Native to exotic plant conversions in central US grasslands have led to highly altered phenology, especially in terms of senescence, and this effect should be considered along with global warming in models moving forward. This conversion will have to be considered in developing estimates of how global change will affect phenology in locations where exotics are present, especially in cases where their abundance is increasing concurrent with climate change. Global change models and policy should consider exotic species invasion as an additional widespread factor behind changes in phenology. This article is protected by copyright. All rights reserved.

Land managers face the challenge of conserving medicinal plants that may be threatened by harvest pressure, often with limited biological information available to inform management decisions. Osha (Ligusticum porteri) is an important medicinal plant whose roots are harvested as an herbal remedy for flu, sore throat, and other illnesses. However, little is known about population structure, root production, or the capacity of osha to recover from harvest in different environmental contexts. We compared osha population structure and root production within a gradient of canopy cover from meadow to forested habitat. We experimentally harvested roots of mature osha plants and recorded osha recolonization of pits created by root harvest. Prior to harvest, the number and percent cover of reproductive plants and the number of flowering stems per plot were higher in the meadow than in forested habitat. Canopy cover had a significant negative relationship to these variables, suggesting that osha populations benefit from increased light availability. Average root weight per plant in meadow plots was three times higher than in forested plots. One year after harvest, the majority of all harvest pits across the canopy cover gradient were recolonized by osha. Our results suggest that osha population structure and root production are significantly influenced by canopy cover, but that plants have a high capacity for post-harvest recolonization under variable light conditions. These results demonstrate the need to account for environmental factors that influence population structure when addressing concerns about the overharvest of wild medicinal plants.

iv CHAPTER 1. GENERAL INTRODUCTION 1 Dissertation Organization 7 References 9 CHAPTER 2. ASSEMBLY HISTORY ALTERS ALPHA AND BETA DIVERSITY, EXOTIC-NATIVE PROPORTIONS AND FUNCTIONING OF RESTORED PRAIRIE PLANT COMMUNITIES 15 Summary 15 Introduction 16 Materials and Methods 20 Results 26 Discussion 28 Acknowledgments 33 References 33 Supporting Information 43 CHAPTER 3. NATIVE SPECIES SEED ADDITIONS DO NOT CAUSE RESTORED PRAIRIE PLANT COMMUNITIES TO SHIFT FROM EXOTIC TO NATIVE STATES 45 Abstract 45 Introduction 46 Methods 48 Results 52 Discussion 53 Acknowledgments 56 Literature Cited 56 Supplementary Information 62 CHAPTER 4. BIODIVERSITY, PHOTOSYNTHETIC MODE, AND ECOSYSTEM SERVICES DIFFER BETWEEN NATIVE AND NOVEL ECOSYSTEMS 64 Abstract 64 Introduction 65 Materials and Methods 68 Results 75 Discussion 78 Acknowledgments 81 References 82 Online Resources 93

The temporal dynamics of ecosystem functioning and services are largely regulated by seasonal patterns, or phenology, of Earth’s biota. Recent evidence suggests that species in exotic-dominated systems may exhibit altered phenology relative to species in native-dominated systems. However, whether phenology of plants and pollinator communities differ between existing novel, exotic-dominated ecosystems and native-dominated systems remains poorly understood. We tested whether exotic- and native-dominated grasslands differed across a growing season for plant production, species and functional diversity, and bee pollinator abundances and diversity in the Northern Great Plains tallgrass prairie region, Iowa, USA. We found that niche overlap and some aspects of phenology in exotic-dominated systems were different from native systems for plants, but not for pollinators. Exotic dominated grasslands peaked earlier in biomass production, and exhibited consistently lower levels of species and functional diversity, including higher levels of proportion of C3 biomass, than native grasslands. These results were related to higher levels of temporal niche overlap in exotic grasslands, where species were similar across the growing season in exotic, but not in native, grasslands. Surprisingly, bee pollinator communities did not differ between exotic and native grasslands despite higher forb:grass ratios in native sites, implying that factors other than exotic-native status may be important in structuring pollinator communities at this scale in highly fragmented landscapes. Our results imply that phenological differences between communities in novel and native grasslands can have important consequences to diversity and ecosystem functioning, and that exotic or native status of plant species should be considered in future studies of phenology.

Biodiversity can be partitioned into alpha, beta, and gamma components, and beta diversity is not as clearly understood. Biotic homogenization predicts that exotic species should lower beta diversity at global and continental scales, but it is still unclear how exotic species impact beta diversity at smaller scales. Exotic species could theoretically increase or decrease beta diversity relative to natives depending on many factors, including abiotic conditions, community assembly history, management, dispersal rates of species, and connectivity among patches. We sampled plant species abundances in 42 novel, exotic- and native-dominated (remnant) grasslands across a latitudinal gradient in the tallgrass prairie region, and tested whether exotic and native grasslands differed in beta diversity at three scales: across sites within the entire biome, across sites within regions, and across locations within sites. Exotic-dominated grasslands differed from native-dominated grasslands in beta diversity at all scales, but the direction of the difference changed from positive to negative as scales went from large to small. Contrary to expectations, exotic-dominated grasslands had higher beta diversity than native-dominated grasslands at the largest scale considered. This occurred because the identity of dominant exotic species varied across the latitudinal gradient, with many exotic grassland pairs exhibiting zero similarity, whereas native-dominated grasslands differed more gradually with distance. Beta diversity among sites within a region was variable, with exotic-dominated grasslands having 29% higher beta diversity than native grasslands in the south and 33% lower beta diversity in the north. Within sites, beta diversity was 26% lower in exotic-dominated than native grasslands. Our results provide evidence that different regional identities and abundances of exotics, and lack of connectivity in fragmented landscapes can alter beta diversity in unexpected ways across spatial scales.

Ecological restorations are predicted to increase in species diversity over time until they reach reference levels. However, chronosequence studies in grasslands often show that diversity peaks after the first few years and then declines over time as grasses become more dominant. We addressed whether bison grazing and seed additions could prevent this decline in diversity. Exclosures that prevented bison grazing were compared with grazed plots over 4 years, and seed additions were conducted inside and outside exclosures to test for seed and microsite limitations. A previous study conducted 4-months post seeding found that local species richness was primarily seed limited, but that grazing could sometimes increase seedling emergence. Here, we tested whether increased seedling emergence led to longer-term increases in the species diversity of the plant community. We found that the seed addition effect grew smaller and the grazing effect grew stronger over time, and that seed additions affected the abundance of added species only when plots were grazed. Grazed plots had higher species diversity and lower biomass and litter buildup compared to non-grazed plots. Our results suggest that moderate grazing by bison or management that mimics grazing can maintain diversity in grass-dominated situations. Our results also emphasize the need to follow seed additions over several years to assess correctly whether seed limitation exists.

Community persistence, or the ability of a community to maintain species composition and diversity through time, is a component of stability that is important to restoration. We ran a biodiversity–ecosystem functioning experiment for three years, and then stopped weeding it for 5–6 years, which allowed us to test whether increased plant species diversity and dissimilarity in height would lead to increased community persistence in the face of high invasion pressure by non-native species. Our approach was unique in that the experiment varied richness (one or four species) and evenness (three levels plus monocultures of the dominant species) using two separate dissimilarity types (having all tall species or having tall and short species combined) in six spatiotemporal blocks. Persistence was quantified as to how well positive productivity–diversity relationships, proportion of planted native species, and species richness remained unchanged over time. Thus, high persistence values indicate low levels of invasion and local extinction. We found that the positive relationship between diversity measures and productivity persisted after cessation of weeding. The proportion of planted species was 32% higher in mixture than in monoculture plots, indicating that monocultures were more heavily invaded by non-native species. Reduced evenness did not affect persistence measures in plots with dissimilar heights, but measures declined linearly with decreased evenness in plots with all tall species. Our results suggest that (1) persistence–diversity relationships are likely to vary with the traits of species becoming rare and going extinct, and (2) it is important to restore higher species diversity in restoration projects to favor the long-term persistence of planted species.

Summary 1. Restorations provide a test of community assembly history theory, and practitioners require information on how assembly history might help to restore diverse native species communities. Variation in community assembly history (historical order of species arrival) is hypothesized to generate beta diversity by producing alternate states, but restorations are hindered because there have been few tests using long-term field experiments. 2. We experimentally altered assembly history of native species into formerly exotic-dominated grassland sites while simultaneously removing dispersal limitation to test whether alternate states or a single equilibrium would develop and whether alternate states would generate varying ecosystem-level effects. Assembly history was altered by varying the identity of earlyemerging species, timing of seed additions after disturbance (early-emerging species added in spring or summer) and priority effects (common 30-species seed mixture added either at the same time or after an early-emerging species canopy developed). The experiment was conducted at two sites that differed in productivity. 3. Altering timing and priority effects during assembly history had large effects on species composition and diversity. On average, diversity was highest, and the proportion of exotic species was lowest in plots seeded in spring and without priority effects. Identity of earlyemerging species did not significantly affect community structure. 4. Differences in species composition affected fuel mass, fire temperatures and peak aboveground primary productivity, key ecosystem processes in tallgrass prairie. 5. Synthesis and applications. Our results indicate that grassland communities can reach alternate exotic- or native-dominated states in uniform environments when perennial exotic species are present. These states were strongly affected by timing of native species arrival and priority effects. Thus, assembly history is a key process that can give rise to beta diversity, and our results suggest that native species should be established early in the restoration process before exotics become fully established.

Summary 1 Grassland restorations often lack rare forb and grass species that are found in intact grasslands. The possible reasons for low diversity include seed limitation, microsite limitation and a combination of both. Native ungulates may create microsites for seedling establishment in tallgrass prairie restorations by grazing dominant species or through trampling activities, but this has never been tested in developing prairies. 2 We experimentally tested for seed and microsite limitation in the largest tallgrass prairie restoration in the USA by adding rare forb and grass seeds in two trials inside and outside native ungulate exclosures. We measured seedling emergence because this stage is crucial in recruiting species into a community. We also measured light, water and standing crop biomass to test whether resource availability could help to explain seedling emergence rates. 3 Ungulates increased light availability for each sampling time and also increased above-ground net primary productivity (ANPP) during summer. 4 Seedling emergence of rare prairie forbs and grasses was consistently greater when we added seeds. 5 Seedling emergence was conditionally greater with a combination of seed additions and grazing, but grazing alone was unable to increase emergence. 6 When ungulates increased seedling enhancement, the mechanism was partially associated with increased water and light availability. 7 Exotic and cosmopolitan weed seedling emergence was not affected by grazing. 8 Synthesis and applications. These results suggest that tallgrass prairie restorations are primarily seed limited and that grazing alone may not be able to increase seedling emergence of rare species without the addition of seeds. Therefore, adding seeds to grassland restorations may increase seedling emergence of rare species, and mimicking effects of grazing may increase emergence when seeds are added.

Species-area relationships and island biogeography theory are commonly used to predict how species richness will decline with fragmentation. There are a variety of largely untested assumptions in these approaches, including the assumptions that populations are distributed uniformly before fragmentation, and that local extinctions are due to effects of small population sizes. If populations are not distributed uniformly, then populations can be abundant locally but rare globally. This would cause extinction rates to be smaller than predicted. We tested extinction theory by developing estimates of the number of plant species that should be present in small tallgrass prairie fragments and then testing the uniformity assumption by partitioning species richness into α (within site) and β (among site) components in Iowa prairies. Many more native prairie plant species were present in surveys of prairie fragments (491) than was predicted based on theory (27-207). A large proportion (75%) of the total species richness was β richness. We suggest that the high proportion of β richness wa sr esponsible for the shallow species-area slopes and the lower than expected number of species losses and that a better understanding of what determines β diversity will improve predictions of fragmentation effects on richness of plants. We also suggest that plants in prairie remnants may be best conserved by protecting different prairie types rather than by protecting a few large areas containing a single prairie type.

Summary 1. We do not know which aspects of community structure and ecosystem processes are restorable for most ecosystems, yet this information is crucial for achieving successful restoration. 2. We quantified three success criteria for 8‐10-year-old grassland plantings in largescale tallgrass prairie restoration (reconstruction) sites relative to three nearby prairie remnant sites. The restoration sites included management of native ungulates and fire, important regulators of diversity and patchiness in intact grasslands. These have not been incorporated simultaneously into previous studies of restoration success. 3. We used the additive partitioning model of diversity, where α is neighbourhood (quadrat) scale diversity, β is accumulation of species diversity across neighbourhoods, and γ is total diversity. We decomposed α into richness and evenness to determine if both were equally restored. 4. The proportion of exotic biomass was similar between the restoration and remnant sites, but the proportion of exotic species and above-ground net primary productivity remained between two and four times higher in the restoration sites. 5. Alpha diversity (Simpson’s 1/D) and richness (S) values were exceptionally high in remnant sites, and approximately twice those of the restoration sites. Alpha evenness was similar between the restoration and remnant sites. 6. Distance per se between quadrats was not related to diversity after accumulated quadrat area was taken into account. Therefore, we may be able to use the additive partitioning model of diversity in areas that differ in size, at least at the scale of this study. 7. Contrary to our original predictions, the proportion of β diversity (1 − D) was approximately twice as high in the restoration sites than in remnant sites, possibly because patches of individual species were larger in the restoration. 8. Synthesis and applications . We have shown that current restoration methods are unable to restore plant diversity in tallgrass prairie. Grassland restoration will be improved if the number of species that co-exist can be increased. New, local-scale restoration techniques are needed to replicate the high levels of diversity observed in tallgrass prairie remnant sites.

Summary During community assembly, early arriving exotic species might suppress other species to a greater extent than do native species. Because most exotics were intentionally introduced, we hypothesize there was human selection on regeneration traits during introduction. This could have occurred at the across- or within-species level (e.g. during cultivar development). We tested these predictions by seeding a single species that was either native, exotic ‘wild-type’ (from their native range), or exotic ‘cultivated’ using 28 grassland species in a glasshouse experiment. Priority effects were assessed by measuring species’ effect on establishment of species from a seed mix added 21 d later. Exotic species had higher germination and earlier emergence dates than native species, and differences were found in both ‘wild’ and ‘cultivated’ exotics. Exotic species reduced biomass and species diversity of later arriving species much more than native species, regardless of seed source. Results indicate that in situations in which priority effects are likely to be strong, effects will be greater when an exotic species arrives first than when a native species arrives first; and this difference is not merely a result of exotic species cultivation, but might be a general native–exotic difference that deserves further study.

Human activities have caused non-native plant species with novel ecological interactions to persist on landscapes, and it remains controversial whether these species alter multiple aspects of communities and ecosystems. We tested whether native and exotic grasslands differ in species diversity, ecosystem services, and an important aspect of functional diversity (C3:C4 proportions) by sampling 42 sites along a latitudinal gradient and conducting a controlled experiment. Exotic-dominated grasslands had drastically lower plant diversity and slightly higher tissue N concentrations and forage quality compared to native-dominated sites. Exotic sites were strongly dominated by C4 species at southern and C3 species at northern latitudes with a sharp transition at 36–38°, whereas native sites contained C3:C4 mixtures. Large differences in C3:C4 proportions and temporal niche partitioning were found between native and exotic mixtures in the experiment, implying that differences in C3:C4 proportions along the latitudinal gradient are caused partially by species themselves. Our results indicate that the replacement of native- by exotic-dominated grasslands has created a management tradeoff (high diversity versus high levels of certain ecosystem services) and that models of global change impacts and C3/C4 distribution should consider effects of exotic species.

Deciding when and how to plant prairie to simultaneously establish native prairie seedlings and prevent weed (non-prairie species) invasion can be challenging. Planting cover crops is an increasingly common management practice for prairie plantings. The idea is based on the assumption that the cover plant will act as a nurse plant to prairie seedlings and will have a positive effect on seedling recruitment by increasing weed suppression. This is predicted to lead to reduced weed biomass and increased prairie establishment in restoration plantings.

Introduction Deciding when and how to plant prairie to simultaneously establish native prairie seedlings and prevent weed (non-prairie species) invasion can be challenging. Planting cover crops is an increasingly common management practice for prairie plantings. The idea is based on the assumption that the cover plant will act as a nurse plant to prairie seedlings and will have a positive effect on seedling recruitment by increasing weed suppression. This is predicted to lead to reduced weed biomass and increased prairie establishment in restoration plantings.

Introduction Prairie restoration projects sometimes fail because of heavy invasion by invasive weeds, especially if they are not intensively managed. Few restoration projects are sampled after the first few years post-establishment, and little is known about what predictors are significant in maintaining restored communities over the very long term. Here, we stopped weeding experimental restoration plots to determine if persistence (that is, remaining unchanged after weeds are allowed to invade) of native prairie in western Iowa was related to planted species diversity.