It is interesting that the low pH did not improve the stability of the anthocyanins, especially compared to a study of single strength elderberry juice cooked at 95 °C at pH 3.5, in which cyn 3-sam had a half-life of 1.87 ± 0.07 h while cyn 3-glu had a half-life of 1.96 ± 0.07 h, values that are more similar to our results. In another study on elderberry anthocyanin stability in a commercial concentrate cooked at 95 °C for 6 h, the overall anthocyanin half-life value was 1.96 ± 0.06 h, which is higher than the values in the current study.145 In another study on elderberry products undergoing thermal processing, the half-life in hours of anthocyanins in pasteurized elderberry juice that contained additives such as sucrose, xanthan gum, potassium sorbate, and ascorbic acid was 26.25 ± 1.49 h at 70 °C, 8.03 ± 0.78 h at 80 °C, and 1.60 ± 0.11 h at 90 °C.146 While the half-life at 70 °C is significantly higher than the values found in the present study at 72 °C, the half-life at 90 °C is similar to what we observed at 95 °C.Because elderberry has predominantly cyanidin-based anthocyanins, protocatechuic acid is typically found as the main degradation product, though phloroglucinaldehyde can also be formed. However, neither protocatechuic acid nor phloroglucinaldehyde were observed in any of the cooked juice samples. Protocatechuic acid dihexoside, raspberries in pots which was tentatively identified in an earlier study of blue elderberry did not increase over the cooking period.

Caffeic acid, a hydroxycinnamic acid increased up to 108.1% of its initial concentration after 2 hours of cooking at 72 °C, and up to 147.1% after 2 hours of cooking at 95 °C. The levelsof caffeic acid were highly variable, with larger standard deviations that the other phenolic compounds. This is a known metabolite of cyanidin-based anthocyanins, and further work investigating the breakdown of anthocyanins in blue elderberry juice into this phenolic acid can elucidate the pathway to this compound. The main flavonols in blue elderberry, rutin and isorhamnetin glucoside, were stable during the thermal processing, retaining 100.5% and 99.3%, respectively, of their original concentration even at 95 °C . The high retention rates of rutin and isorhamnetin glucoside match literature reports for the thermal stability of these compounds, which show that rutin has a strong thermal stability at acidic pH. More than 80% of the starting concentration was retained after five hours of cooking at 100 °C at pH 5. Our results do not agree with another study in which rutin had an activation energy 107.3 kJ/mol, and the half-life values at 70 and 90 °C were 19.25 and 1.99 h, respectively; however, the rutin was in an aqueous solution at pH 6.6.149 Other compounds present in blue elderberry juice, in addition to a lower pH, could cause synergistic effects to improve stability of rutin in the present study.

Limited information on the thermal stability of isorhamnetin glucoside was found, though a study of black currant juice stability found that during long-term storage at room temperature and at 4 °C, isorhamnetin glucoside concentrations did not change significantly during the 12-month period. In the same study, rutin did not change significantly during storage. The main phenolic acid in blue elderberry juice, chlorogenic acid, was also thermally stable. This result was unexpected, as another study on the thermal stability of chlorogenic acid in a complex with amylose showed a significant decrease in content after 10-15 minutes, depending on the temperature. Their results also showed that a 10 °C increase in temperature results in a 2.5-fold increase in the rate of degradation of chlorogenic acid. It can be beneficial to maintainlevels of chlorogenic acid in anthocyanin-rich matrices, as shown in black carrot extract where chlorogenic acid increased absorbance of cyanidin-based anthocyanins at pH 3.6 and 4.6 due to intermolecular co-pigmentation. Overall, our results show that blue elderberry juice behaves similarly to anthocyanin-rich matrices, in that longer processing at higher temperatures degrades anthocyanins. The two main anthocyanins in blue elderberry, cyn 3-sam and cyn 3-glu, behaves similarly during processing, degrading at about the same rate at 72 °C and 95 °C. Furthermore, the other major phenolic compounds like rutin, isorhamnetin, and chlorogenic acid, were highly stable and can withstand the thermal processing. Our study into the effects of thermal processing on the phenolic composition and cyanogenic glycoside content in blue elderberry juice showed that the main anthocyanins present degrade faster at higher temperatures but other important phenolic compounds like rutin and isorhamnetin 3-glucoside are more thermally stable, retaining over 90% of their original concentrations even after two hours at 95 °C.

Furthermore, neoamygdalin and sambunigrin were measured in the blue elderberry juice, which were in lower concentrations compared to European and American elderberry. Globally, native pollinator species are facing both range contractions and population declines due to a combination of threats including; habitat loss, disease, agricultural intensification and global climate change . While we still do not have sufficient long term data to understand the extent to which pollinator declines will impact the plants that rely on them for reproduction, some studies suggest that plants and pollinators are facing concurrent declines . Still, recent network modeling work based on pollination networks suggest that plant communities will be highly resilient to linked extinctions when faced with pollinator declines . This is because pollination networks have an asymmetric structure and because these networks are dominated by generalist interactions. Generalism is thought to add functional redundancy. That is, when a pollinator is lost from the community there is another that can fill its functional role. Simulations of pollinator species losses suggest that the asymmetric, generalist-dominated structure of pollination networks should provide extreme resilience to extinctions of either plants or pollinators . These predictions of resilience are promising, but have not been tested empirically. My PhD research is focused on understanding the implications of pollinator species losses.Global pollinator declines are ongoing , leading to concerns about the functional impacts of pollinator species losses on both pollination-dependent native plants and crop production . Network-based simulation models of pollinator species losses, however, predict that major functional impacts on plant species persistence will not typically result until many, or even most, pollinator species are lost from a system , but these results have not been tested empirically. These predictions are based in part on redundancies in pollination networks: most plant species are visited by several pollinator species, contributing to the robustness of such networks . This robustness, however, is dependent on the implicit assumption that the functional roles of pollinator species are static. If interspecific interactions can dynamically alter species’ functional contributions—such as the efficacy of a particular plant-pollinator relationship— pollinator species losses may have greater cascading impacts on plant communities than predicted by current models. Such dynamic changes in species functional roles are predicted by the ecological literature on interspecific competition and specialization, but have not been studied in the context of pollination or other ecosystem functions and services. Competition between species has long been known to affect specialization in a wide range of taxa and ecosystems over both evolutionary and ecological timescales . When interspecific competition increases, each species in a system tends to specialize ecologically. By contrast, when intraspecific competition increases, blueberries in containers growing individuals within that species tend to become more ecologically generalized. When considered over evolutionary time, this process can lead to niche differentiation , resulting in static ecological complementarity among species, a well-documented mechanism for positive biodiversity-ecosystem function relationships . Over ecological timescales, however, interspecific competition can lead to dynamic specialization via phenotypic plasticity, either behavioral or morphological.

In the case of pollination, short-term pollinator foraging specialization on particular plant species, or “floral fidelity”, is critical because transfer of conspecific pollen must occur in order for fertilization to take place. Floral fidelity may be shaped by interspecific interactions because most pollinator species are generalists with labile foraging patterns. Despite the potential importance of dynamic specialization, its contributions to ecosystem services and functions has not been considered to date.Here, we assessed the impacts of single-pollinator species losses on dynamic specialization and pollination function using novel field manipulations. Our manipulations temporarily and non-destructively removed the most abundant bumble bee species from field plots using targeted aerial netting. The identity of the target species varied between plots, and we manipulated 6 Bombus species, or more than half of the 11 species in the system. We assessed each individual plot in both a control and a manipulated state, allowing us to hold the plant community constant within our comparisons. We assessed how dynamic specialization of bumble bees changed between the control state and the manipulated state of each plot, and followed the linked changes through the steps of the pollination process in terms of bee pollen carriage, deposition of pollen on floral stigmas, and finally plant reproductive function, i.e. seed output . We tracked the pollination process in a larkspur, Delphinium barbeyi , an abundant wildflower that is visited by at least 10 of the 11 bumble bee species in the system.Rates of floral fidelity, proportions of conspecific pollen carriage and deposition, and seed production significantly decreased in the manipulated state—with a single pollinator species removed—relative to the control state. We assessed floral fidelity on a per-plant-visit basis and on a per-bee basis . On a per-plant-visit basis, foraging movements between individual plants of different species increased by an average of 156% in manipulated plots relative to controls, based on observation of >23,500 between-plant foraging movements in 736 individual bumble bees . On a per-bee basis, the percentage of individual bees visiting only one species of plant within a single foraging bout decreased from 77.7% to 66.4% in the control relative to the manipulated state of each plot in the same 736 individual bees . Patterns of pollen carriage also reflected decreased floral fidelity: bumble bees in the manipulated state carried 17.5% more mixed-species pollen loads relative to controls . The proportion of conspecific pollen deposited on D. barbeyi stigmas concurrently decreased from 61% to 56% in control vs. the manipulated state . These changes in specialization behavior, pollen carriage, and stigmatic deposition were ultimately reflected in decreased ecosystem function, i.e. a significant reduction in seed production in D. barbeyi in the manipulated relative to control state of each plot . Based on GLMM model coefficients and mean Bombus species richness and abundance, single-pollinator species removals reduced mean seed count per flower by 32.0%.Our results also provide support that the effects of the manipulations on plant reproductive function were driven by differences in pollinator species richness, rather than changes in abundance. The single pollinator species removal manipulations reduced Bombus relative abundance on average, though the difference was marginally non-significant . Individuals of non-target Bombus species could freely enter the plots, and abundance effects were highly variable . In 7 of the 20 sites, Bombus abundance was greater in the manipulated state relative to the control , allowing us to statistically control for changes in Bombus abundance due to the manipulations. Bumble bee abundance was not significantly related to either of the two floral fidelity outcomes or to pollen carriage . The relationships between Bombus abundance and both stigmatic pollen deposition and seed set, while statistically significant, were negative with small model coefficients , i.e. indicating that increased bumble bee abundance was associated with slightly lower conspecific pollen deposition and seed production. Thus, the pattern of reduced ecosystem function in the manipulations is not likely driven by abundance changes.pecies, despite the fact that other potentially effective pollinator species remained in the system. This empirical finding contrasts with prevailing predictions of network robustness to pollinator species losses, based on simulation models that do not account for changes in the efficacy of particular plant-pollinator relationships. The reduction in plant reproductive function that we observed is associated with dynamic changes in pollinator behavior, through the steps shown in Fig. 1. When we removed an interspecific competitor, remaining pollinators broadened their foraging strategies to include flowers that the removed species had previously focused on. This dietary broadening within a single foraging trip means that remaining pollinators show less fidelity to particular plant species.