Horticulture

Additionally, relationships between traits and evolutionary history also result in mutual correlation among traits; thus, we also constructed a Spearman rank correlation matrix of traits . In constructing nested linear models and the correlation matrix, lecty was converted from a categorical variable into a quantitative variable corresponding to diet niche breadth to aid in model fitting.Across two years of sampling, we found that study plots in fragments harbored bee assemblages with reduced plot-level functional diversity and distinct functional composition compared to those in reserves. Changes in functional diversity and composition were closely related to declines and shifts in taxonomic diversity and composition. While we found strong evidence for non-random patterns of species loss, such patterns of loss was insufficient to cause landscape-level taxonomic or functional homogenization in the fragments. Null model analyses and correlational analyses also demonstrate that the loss of bee functional diversity can be explained by loss of bee taxonomic diversity. Taken together, these findings suggest that ecological filtering contributes to the restructuring of bee assemblages, large plastic gardening pots but is not the main driving force of bee diversity loss in habitat fragments in our system.

The strongest support for the importance of ecological filtering in our system is the detection of multiple indicator species and functional groups that appear particularly susceptible to fragmentation, typical of “winner-loser” dynamics found in modified landscapes . Also typical of “winner-loser” dynamics, we found a number of species that are present at all study plots, most of which are eusocial species in the tribe Halictini, which are known to be tolerant of habitat fragmentation . However, unlike systems where small numbers of “winner” taxa or functional groups dominate modified landscapes , indicator analyses revealed no such “winner” species or functional groups associated with fragments. Our finding only indicator taxa associated with reserves suggests that ecological filtering indeed leads to the exclusion of certain “loser” taxa and functional groups from fragments, but not to such an extent that the bee assemblages become numerically dominated by groups of disturbance-tolerant species that thrive in altered habitats. In fact, the loss of “loser” taxa seems to largely underlie the detected directional shifts in both taxonomic and functional measures of assemblage composition; simply removing the 12 indicator species from the analyses nullifies the significant differences detected between reserves and fragments with respect to both taxonomic and functional composition .

Evaluating differences between bee faunas in reserves and fragments one trait at a time revealed several differences between reserves and fragments, but only two that remained statistically significant after correction for multiple comparisons . Preferential loss of specialists in modified environments has been documented in many taxa , including bees . The increased relative abundance of late-season active bees observed in the present study has also been reported in at least one other system in which bees in modified landscapes have enhanced access to anthropogenic sources of floral resources during periods of relative resource dearth . In our system, it is likewise plausible that late-season bees in fragments are able to thrive by foraging on floral resources in the irrigated urban matrix surrounding fragments. The increase in average range size of bees inhabiting fragments also reveals the role of ecological filtering in structuring bee assemblages our system. Range size is not a functional trait per se, but it does serve as a proxy for an important ecological function that remains difficult to quantify: overall niche breadth . While many studies on bees focus on lecty as the main metric for niche breadth , selectivity of nesting substrates , phenological flexibility , and physiological tolerance to abiotic conditions may all influence how bee species respond to the addition of novel ecological filters. Our results suggest that bees in fragments tend to be those that are capable of surviving in a greater number of ecological contexts compared to bees in reserves, consistent with the view ecological filters present in habitat fragments exclude species that are more narrowly adapted to the unique local ecosystems.

Such replacement of endemics by geographically widespread species has been observed in other systems impacted by habitat alterations , and may be an important force driving reductions in ecological complexity across large spatial scales. Given that bee assemblages in fragments exhibited strong reductions in both taxonomic and functional alpha diversity as well as distinct taxonomic and functional composition compared to reserves, it is noteworthy that reserves and fragments did not differ with respect to either taxonomic or functional beta diversity. Reduced beta diversity is associated with biotic homogenization, which is a hallmark of ecological filtering resulting from anthropogenic disturbance . Biotic homogenization resulting from land use change has been found across many taxa , including pollinators . However, unlike other systems in which anthropogenic impact is dominant and pervasive, such as in cases where intensive agriculture generated highly simplified landscapes , the habitat fragments we selected in our study were comparable to our natural reserve sites with respect to both the diversity and the composition of native, insect-pollinated plant assemblages, at least at the scale of our one-hectare study plots . Thus, given that our fragment plots retained relatively intact plant assemblages, it is perhaps unsurprising that bee assemblages therein had not converged to a subset of taxa that thrive in altered habitats . As with the findings of , robust beta diversity among fragments may result from underlying heterogeneity in the habitat characteristics of our fragment plots. Taxonomic and functional diversity are often positively related to each other , but the two measures of diversity are related to each other in complex ways and may be independently impacted by habitat modifications . These complex relationships may explain our null model analysis, wherein the reduction in functional diversity in fragments did not differ from expectation under stochastic species loss despite our detecting multiple “loser” functional groups that suffer declines in fragments. While the parallel declines in taxonomic and functional diversity we detected in fragments via both null model and correlation analysis may indeed indicate stochastic loss of species , such a pattern could also arise from non-random loss of species whose functional traits have dispersions comparable to those lost due to random removal of species in the null model. Our finding that the “loser” species and functional groups associated with reserves varied with respect to every functional trait measured lends support to the latter mechanism. Alternatively, the apparent non-uniformity in the functional traits of “loser” taxa may result from our not measuring some other functional traits that may be shared among these taxa. For example, if dispersal is the main driver of bee assemblage composition in fragments, a functional trait that strongly influences the likelihood of dispersal across the urban matrix may be largely responsible for interspecific variation in likelihood of local extirpation from fragments. Irrespective of the mechanism underlying the parallel declines in taxonomic and functional diversity, our null model and correlational analysis results suggest that in our system, managing habitats in such a way as to preserve taxonomic diversity may be an effective way to preserve functional diversity . We uncovered significant phylogenetic conservatism in the functional traits we measured , large plastic growing pots which likely contributed to the numerous correlations detected among traits , a pattern also reported in other studies involving bee functional traits .

Given that phylogenetic conservatism in functional traits can shape the ecology and distribution of bee species in a landscape , our findings must be interpreted in the context of fragmentation impacting bees at the level of higher taxa. However, since analyses at the level of genera yielded qualitatively similar results , the overall patterns we report are unlikely to be driven by a few species-rich groups that respond especially strongly to fragmentation. The detection of indicator species belonging to three families and indicator functional group members belonging to five families also suggests that impacts of fragmentation are not limited to certain clades of bees. Phylogenetic relationships among bee taxa are a subject of ongoing research, even at the level of higher taxa . Once accepted phylogenies become available for bee taxa occurring in our system, it would be instructive to quantify the extent to which evolutionary relationships among taxa contribute to our findings, and the implications fragmentation may have on the evolutionary trajectory of bee faunas as time progresses.The maintenance of both taxonomic and functional beta diversity in our studied fragments argues for the preservation of each individual fragments of CSS habitat, despite the fact that fragments as a whole share the absence of sensitive “loser” bee taxa and functional groups. Our results suggest that each fragment preserves its own distinctive subset of the bee faunas formerly present in the regional species pool, and thus by extension, their ecological interactions with other taxa such as plants, parasitic or commensal invertebrates , and microbes . High levels of heterogeneity in assemblage composition among fragmented habitat remnants have also been documented in other systems ; in such systems, the cumulative species pool of compositionally divergent fragments may equal or exceed the species pools of unfragmented habitat. Beta diversity as a result of habitat heterogeneity is a strong driver of local and regional diversity of pollinators and organisms in general . In our system, bee faunas occupying habitat fragments embedded in a heterogeneous landscape do not exceed or equal those in larger natural reserves with respect to taxonomic or functional diversity, but nevertheless represent valuable units of conservation that may each exhibit unique community-level evolutionary trajectories with time if properly preserved. In fact, of the 216 species collected in the study , 40 were unique to fragments , while 74 were unique to reserves . That said, the decrease in plot-level functional diversity in habitat fragments still represents a conservation challenge with respect to both the functionality and the resilience of bee faunas , highlighting the importance of preserving large, intact areas of scrub habitat.We demonstrated that ecological filtering in fragmented scrub habitats caused shifts in the taxonomic and functional composition of bee faunas as a result of a loss of sensitive bee taxa and an increase in the relative abundance of geographically widespread bee species. However, filtering was not sufficiently strong to reduce functional diversity beyond that expected under random species loss, and bee faunas in fragments retained taxonomic and functional beta diversity among plots. Future studies that can quantitatively partition the relative contribution of deterministic and stochastic processes in driving taxonomic and functional diversity loss will shed light on the factors influencing community reassembly in structurally intact but isolated fragments of well preserved natural habitat.Animal-mediated pollination of angiosperms represents a vital ecosystem function in terrestrial ecosystems . Thus, reported declines in pollinator abundance and diversity worldwide could harm the integrity of terrestrial ecosystems. For this reason, documenting how environmental change impacts the structure and function of plant-pollinator interactions has been identified as an important research priority . In the last two decades, the bipartite network approach has become widely favored for examining interactions between communities of flower visiting animals and plants. To construct plant-pollinator interaction networks, researchers document the frequency with which each pollinator species visits each plant species within a predefined area. The resulting topology of interaction patterns provides information regarding the manner in which species are connected to one another, and the number of interactions documented between two species is often used as a surrogate for the strength of the relationship between the two putative mutualists with respect to pollination services or food provision . While studies on the structure of plant-pollinator interaction networks provide no direct information on the fitness of organisms involved, general patterns in network structure across plant and pollinator communities have shed light on the function of these networks. For example, nestedness and asymmetry , two properties common to most networks studied, result from the presence of groups of numerically abundant, ubiquitous generalist species that interact with large numbers of partner taxa. Having such generalized species at the “core” of networks may cause the ecological function of these networks to be robust to the loss of species and habitat . However, if network structure indeed predicts the resiliency of ecological relationships between plants and pollinators, then perturbations to network structure resulting from anthropogenic impacts may have strong consequences on ecological function Given the link between network structure and ecological function, a number of studies have investigated how plant-pollinator interaction networks are impacted by different sources of anthropogenic impact such as habitat fragmentation , land use intensification , grazing , and biological invasions .

Another issue is the final products of sulfate and nitrate reduction are toxic to animals at higher concentrations. Lastly, fumarate-reduction uses hydrogen to produce succinate and eventually into propionate, which can be utilized by the animal compared to CH4 .The greenhouse gases CH4 and CO2 along with nitrous oxide , water vapor and fluorinated gases are responsible for trapping radiation emitted from the surface of the planet and preventing its dissipation into the atmosphere. The trapping of radiation leads to an increase in the planet’s temperature, causing the “greenhouse effect”. Carbon dioxide is the most abundant greenhouse gas emitted globally at 65% followed by CH4 and N2O at 16 and 11%, respectively . Even though CH4 constitutes a smaller relative abundance of greenhouse gas and has a shorter life span , CH4 has 28 times the global warming potential of CO2 over a 100-year period. It is estimated that agriculture globally contributed 5.5 gigatons of carbon dioxide equivalent per year with enteric fermentation contributing 2.1 GtCO2eq/year in 2010 . This does not include CO2 emissions as it is considered neutral from carbon fixation.

When plants and crops are grown, they take up the CO2 in the atmosphere, and are released again when they are degraded, square pot plastic thus not creating a net change in CO2. Enteric fermentation contributes to 32% of global anthropogenic CH4 while also representing 2-12% loss of gross energy as the animal cannot use it . As fore mentioned, there are other pathways from carbohydrate fermentation that can act as alternative hydrogen sinks such as acetogens and propionate producers. Feed composition plays a big part in ruminal fermentation, with diets containing higher soluble carbohydrates promotes more propionate production whereas diets with higher fiber produce more CH4 alongside acetate . Many plant products also contain bio-active compounds such as plant secondary metabolites which contain antimicrobial properties that reduce or inhibit CH4 production. Plant secondary metabolites contribute to their survival via defense against external threats such as pathogens, grazing herbivores, and environmental conditions. These bio-active compounds are also responsible for the unique aroma and taste of different species of plants.

Plant secondary metabolites are classified into different families according to their chemical structures: phenols, organosulfur compounds, terpenes, and alkaloids.Plant phenolic compounds show promising results for reducing enteric CH4. Phenols are organic compounds consisting of an aromatic ring binding to at least one hydroxyl group. Polyphenolics are composed of multiple phenols which determine their chemical properties and comprise over 8,000 different polyphenolic compounds. These polyphenolic compounds contain antimicrobial properties that either directly inhibit methanogens or indirectly reduce methanogen activities by reducing fiber degradation from other microbes thus reducing overall fermentation and substrates. Date palm leaves are byproducts generated from date production, which are an important food staple in regions with little to no rain fall. Even though they are not fit for human consumption, DPL can be used as a feed for ruminants as they are high in fiber and contain polyphenolic compounds. When replacing berseem hay with DPL in-vitro,there was a linear decrease in total gas production, CH4 and CO2 . Date palm leaves had higher fiber content which requires longer time to degrade, resulting in the decrease in fermentation. Thus, DPL was also ensiled for 45 days with fibrolytic bacteria to increase the soluble fiber available for rumen fermentation.

When the treated DPL replaced berseem hay at 100%, CH4 production was decreased to 89.2mL/g degraded DM compared to 111.3mL/g degraded DM, respectively . Additionally, in diets containing treated DPL, there was an increase in total VFA concentrations compared to the berseem hay . Date kernels are another byproduct from date processing, containing high nonstructural carbohydrates and can be used as a concentrate in ruminant diets, which supplies energy for the animal . They also contain a variety of phenolic compounds that can inhibit ruminal microbe activities. In an in-vitro study that replaced maize with DK from 25 to 100%, up to 39% of CH4 production was suppressed when DK completely replaced maize . Date kernels versus corn had a higher amount of phenol content at 217g/kg DM to 36g/kg DM, respectively. It was found that the most abundant phenolic compound was protocatechuic at 58% followed by p-hydroxybenzoic acid at 16% in date kernels .Tannins are a subgroup of polyphenolic compounds, subdivided into hydrolysable tannins or condensed tannins . Hydrolysable tannins are characterized by a carbohydrate molecule, most commonly glucose esterified by a phenolic acid. Depending on the phenolic acid, they are grouped into either gallotannins or ellagitannins. Gallic acid is the predominant phenolic acid in gallotannins , whereas ellagitannins contain the hexahydroxydiphenoyl ester and are modified into ellagic acid, a dimeric form of gallic acid during metabolism . When HT are broken down by tannin-digesting microbes, gallotannins are transformed into gallic acid and further into pyrogallol. Pyrogallols are toxic to bacteria as they inhibit growth through interactions with proteins or polysaccharides found on their cell walls or directly with the lipid membrane . However, the exact mechanism in which pyrogallol interacts with rumen microbes is poorly understood. Tannins can be found in pomegranate peels with HT making up 90% of the tannin content . It is estimated that the pomegranate industry generates over 1.47 tons of byproducts in the form of peels yearly. In an in-vitro study that replaced hay with pomegranate pomace at a rate of 500g/kg of DM, CH4 production decreased by 28% over a 24 h fermentation period . Even though CH4 production significantly decreased, adding pomegranate pomace did not alter VFA production. This shows that pomegranate pomace can be a good candidate for in-vivo studies to potentially reduce CH4 reduction without compromising the productivity of the host.Condensed tannins are polyphenols that are made up of two or more flavan-3-ols compounds and are not water soluble. They bind directly to proteins and polysaccharides, and in doing so reduce the digestibility of feed. With the reduction of fermentation, there are less substrates available such as CO2 and H2 for methanogenesis. Mango wet waste such as peels and seeds are also a large part of byproduct waste from mango consumption. Though inedible for humans, they still contain high amounts of carbohydrates, protein, and fats that can be used in animal feed. When replacing yellow corn with mango seed kernels in-vitro from 5-20%, there was a linear decrease in CH4 production with no negative effects on VFA production . On the contrary, there was an increase in VFA production in the 10 and 20% treatment, which may be due to seed kernels being more digestible than yellow corn. Mango leaves are also byproducts generated from the mango industry following the pruning process of mango trees. They contain high amounts of tannins that can be used in place of traditional rough ages. When replacing wheat bran with deciduous mango leaves in-vitro at 40%, square pots for plants there was a significant decrease in gas production . Mango leaves contained a higher amount of tannin compared to wheat bran . The lower gas production and fermentation can be attributed from the tannin present but also other compounds such as ether extract and fiber. Since CT decrease digestibility and fermentation, it should only be given to animals in regulated amounts as too much can be detrimental to the health and production of the host.Terpenes are naturally occurring hydrocarbons built from two or more units of isoprene, a volatile 5-carbon compound, and are responsible for plants’ unique aroma, taste, or color. Terpenes can range in size from monoterpenes to polyterpenes of as many as 15,000 isoprene units. Terpenoids, also known as isoprenoids, are modified terpenes which contain oxygen. They also contain functional groups modified by the addition of oxygen atoms.

Terpenoids are the largest class of plant secondary metabolite with over 60,000 identified structures. Olive leaves from the pruning process of olive production contain high amounts of bio-active compounds that can exert antimicrobial effects. When replacing timothy hay with OL by 5% in-vitro, there was a 34% decrease in CH4 production after 12 h .However, the decrease in CH4 production was only temporary as there was no difference in CH4 production at 24 h. The temporary decrease may be due to the lower digestibility from the high fat content and terpene-based polyphenols present in OL. Terpene-based compounds exhibit antimicrobial properties by disrupting cellular membranes . There was also a significant decrease in bacteria, methanogenic archaea, and fungi after 24 h of fermentation in the OL group, though there were no differences in VFA production between the two groups. Further studies are required to understand the long-term effects of OL on rumen microbes and their fermentation in longer in-vitro studies and in-vivo.Essential oils are composed mainly of mono- and sesquiterpenes and are highly concentrated in plants. Essential oils are extremely volatiles and aromatic compounds which plants utilize for a variety of purposes, such as attracting or repelling insects. Essential oils are also used by humans for a variety of purposes, such as ingredients in cosmetics, aromatherapy, or as antimicrobial agents. There are many methods of extractions such as steam distillation, solvent extraction, hydro-distillation, and supercritical CO2 extraction . Essential oils have been shown to exhibit antimicrobial activity but is highly dependent on the presence of other compounds such as phenols . Orange leaves generated from pruning of orange trees contain high amounts of essential oils but is highly dependent on their geographical locations . They can be used as a feed alternative to traditional forages in ruminant diets. However, when OL replaced alfalfa hay in the ration for lactating goats, there was a decrease in dry matter intake but no significant differences in milk yield between the two groups . Between the two groups, the OL had a decrease in CH4 by 3.8g/kg of milk produced . Though they could not identify the compounds directly responsible for the suppression of methanogenesis, it is speculated that the essential oils and tannins inside their leaves may have been responsible. Grapefruit peels also contain citric essential oils that have antimicrobial and antioxidant properties that can be effective in inhibiting methanogenesis. When CEO from grapefruit were added in-vitro with rumen fluid from 6 male Hu sheep at 0.8mL/L and 1.6mL/L, there was a negative relationship for CH4 production as dosage increased . However, VFA production also declined as CEO do not target a specific group of microbes but rather reduce overall fermentation activity. An in-vivo study was conducted alongside the in-vitro study and found that when CEO was added at a rate of 0.8mL/L of rumen fluid, there were no changes in DMI, body weight , and average daily gain . Citric essential oils from grapefruit peels were mainly comprised of D-limonene at 80% followed by B-pinene at 5.52%. Dlimonene is a monoterpene that has been shown to be an effective antimicrobial compound due to its disruption of cellular membranes .Sulfur is an essential nutrient for the growth and development of plants. Glucosinolates are secondary metabolites that contain glucosides linked with sulfur within their structures. Glucosinolates are mainly found in cruciferous vegetable such as broccoli, cabbages, kale, and other leaf plants . There have been around 200 types of glucosinolates identified with differing structures dependent on plant type, environmental factors, and stage of growth . Glucosinolates are important in the plant defense system as it is rapidly hydrolyzed by the enzyme myrosinase into mainly isothiocyanates when the plant tissueis mechanically damaged resulting in a pungent sulfuric odor that acts as a deterrent to herbivores . Stalks and leaves from broccoli are usually left unutilized as they are not consumed by humans. Belonging to the Brassicaceae family, alongside other crops such as rapeseed, cauliflower, and turnip can have differing effects on rumen fermentation dependent on their vegetive stage. When tested in-vitro using rumen fluid from two sheep, vessels containing only broccoli substrate had a 35% lower CH4 production compared to treatments containing only rapeseed . Though further studies need to be conducted on broccoli when compared to traditional feeds, these results demonstrate that different cultivars under the same family can have differing effects on rumen fermentation.Polyunsaturated fatty acids are fatty acids containing two or more double bonds as part of their hydrocarbon chain. They are found in some animal sources such as fish but are more commonly found in plant-based sources such as nuts and seeds.

Accordingly, a synthesis of 600 fields from 41 crop systems showed that only two of the 68 most frequent pollinators globally were specialist species: the weevil Elaeidobius kamerunicus pollinating oil palm and the squash-bee Peponapis pruinosa pollinating pumpkin .Because of differences in species functional traits, greater pollinator richness can lead to foraging complementarity or synergy, improving the quantity and quality of pollination and therefore increasing both the proportion of flowers setting fruits and product quality . Across crop species, insects with contrasting mouth part lengths may be needed for the pollination of flowers not only with easily accessible rewards but also with rewards hidden at the bottom of a tubular corolla . Within a crop species, social and solitary bees visited flowering radish plants at different times of day, suggesting temporal complementarity among these pollinator groups . Flower visiting behavior also differs among pollinators of different body sizes, and visits by a range of differently sized pollinator species increase pumpkin pollination. In addition to functional traits, square pot plastic interspecific differences in response traits to climate and land-use change can increase resilience of pollination services.

The role of diverse assemblages of wild insects in crop pollination is also evident from recent global analyses. Worldwide, incomplete and variable animal pollen delivery decreases the growth and stability of yields for pollinator-dependent crops . This lower yield growth has been compensated for by greater land cultivation to sustain production growth . The consequent reduction in natural areas within agricultural landscapes decreases the richness and abundance of wild pollinators, including bees, syrphid flies, and butterflies , further diminishing crop pollination . A possible solution to this “vicious cycle” is to increase pollinator abundance through single-species management, most commonly European honey bees , which are not greatly affected by isolation from natural areas . However, increasing the abundance of one species may complement but not replace the pollination services provided by diverse assemblages of wild insects, and wild insects pollinate some crops more efficiently than honey bees . Moreover, during the past 50 years, the fraction of animal-pollinator dependent agriculture and the number of managed honey bee hives have increased 300% and 45%, respectively, and honey bees have suffered from major health problems such as colony collapse disorder . All of these factors point to the potential benefit of practices that boost the species richness and abundance of wild pollinators.

Indeed, richness and visitation rate of wild pollinators are strongly correlated across agricultural fields globally . Therefore, practices that enhance habitats to promote species richness are also expected to improve the aggregate abundance of pollinators, and vice versa .Below we describe practices that diversify and improve the abundance of resources for wild insects outside the crop field, without affecting crop management. Practices are ranked from less-to-more required area, with practices covering less area likely to be less costly . Nesting resources – such as reed internodes and muddy spots for cavity nesters, and bare ground for soil nesters – can be enhanced at crop field edges without affecting much of the crop area. Although providing such resources can promote the recruitment of certain bee species , evidence of its effects on crop yield is lacking . Hedgerows and flower strips are woody or herbaceous vegetation, respectively, planted at the edge of a crop field, and generally covering only a small area. If appropriate plant species are chosen and adequately managed through time , hedgerows and flower strips can provide suitable food and nesting resources for, and enhance species richness and abundance of, bees and syrphid flies . These practices also enhance pollinators in adjacent fields – rather than simply concentrating pollinators at dense flower-rich regions – and therefore increase crop yield .

Regional programs that augment the quality and availability of seeds from native flowering plants are important for the success of these practices . Conserving or restoring natural areas within landscapes dominated by crops often provides habitat for wild pollinator populations . In addition, pollinators depend on various types of resources , which are difficult to provide in ways other than by enhancing natural areas. Consequently, these areas also enhance pollination services for nearby crops . Enhancing farmland heterogeneity increases pollinator richness because plant species provide complementary resources over time and space, and insect species use different resource combinations . Also, insects usually require resources for periods longer than crop flowering . In fact, a synthesis of 605 fields from 39 crop systems in different biomes found that diversity of habitats within 4 ha enhanced bee abundance by 76% as compared with bee abundance in monoculture fields . Smaller crop fields increase land-use heterogeneity, and also benefit pollinators because most species forage at distances less than 1 km from their nests . Thus, crops in small fields are more likely to benefit from pollinator enhancements such as nearby field margins and hedgerows . Indeed, pollinator richness, visitation rate, and the proportion of flowers setting fruits decreased by 34%, 27%, and 16%, respectively, at 1 km from natural areas across 29 studies worldwide .In contrast to off-field methods that can be ordered from smaller to larger scale , on-field practices are all applied at a similar spatial scale, ie that of the crop field. Here we discuss practices that reduce the use of insecticides and machinery, enhance the richness of flowering plants, and require greater effort because of changes in the crop species or system . Reducing the use of synthetic insecticides that are toxic to pollinating insects should provide an important benefit . For example, in South Africa, insecticides adversely affected pollinators, impairing rather than enhancing mango yield . Insecticides with low toxicity to pollinators, with non-dust formulations, applied locally through integrated pest management practices, and applied during the non-flowering season are less likely to be detrimental to pollinators than highly toxic, systemic insecticides that are broadly sprayed from airplanes . No-tillage farming may enhance populations of ground nesting bees given that many species place their brood cells <30 cm below the surface . Tillage timing, depth, and method probably have differential impacts on pollinators and pollination, but further studies are required to verify this expectation . Similarly, flood irrigation may be detrimental in comparison to drip irrigation because of the increased likelihood of flooding pollinator nests but, particularly in arid systems, irrigation in general can promote wild-insect abundance through higher productivity of flowering plants or by making the soil easier to excavate . Enhancing flowering plant richness within crop fields can benefit pollinator richness and crop pollination, as demonstrated for mango and sunflower in South Africa. Similar results were found for wild plants within watermelon and muskmelon fields in the US . In Ghana, banana intercropping with cocoa boosted pollinator abundance and cocoa pod set . A diverse set of flower species with different phenologies is likely to increase resource stability for pollinators and thus the resilience of pollination services. Herbicides and mowing can negatively affect pollinators by reducing floral resources provided by weeds , square pots for plants but can be useful for reducing the abundance of invasive grasses that could otherwise displace native flowering plants . Organic farming combines some of the practices described above and can enhance wild pollinator populations in comparison to conventional farming , probably because of the absence of synthetic insecticides and/or greater non-crop floral resources.

Farmland heterogeneity can also be increased by organic management practices, which account for less than 1% of global agriculture . When the extent of organic farming was expanded in a German agrolandscape from 5% to 20%, bee richness rose by 50%, while the density of solitary bees and bumble bees increased by 60% and 150%, respectively . Pollination-related benefits of organic practices were also found for strawberry in Sweden and canola in Canada . Sowing flowering crops, instead of crops that do not offer floral resources for pollinators, may enhance wild pollinators in heterogeneous landscapes . In western France, solitary-bee richness and abundance were higher in margins of canola fields than in fields of other crops . In the UK, bumble bee abundance was higher in areas adjacent to bean fields than to wheat fields but only during crop flowering , suggesting a short-term behavioral response to flower abundance rather than a long-term population enhancement. Similarly, in Germany, canola improved bumble bee early-colony growth but not whole-season sexual reproduction , and greater land cover of mass-flowering crops increased the number of bumble bee workers but not colony numbers . Therefore, although crops can provide abundant resources, the short duration of floral availability, the low diversity of resources, the application of insecticides, and the presence of tillage may limit the capacity of one crop species to support wild pollinator populations on its own . Furthermore, large monocultures of flowering crops can suffer from pollination deficit and trigger indirect negative effects on pollinators . Sowing crops that bloom in different periods may therefore increase wild-insect populations; in Sweden, bumble bee reproduction was improved in landscapes with both late-season flowering red clover and early-season mass-flowering crops . Moreover, managing crop phenology to better match the availability of efficient pollinators should enhance pollination, but we found no studies on this practice .The effectiveness of pollinator-supporting practices is influenced by interactive effects between large and small scale factors. For example, the effects of landscape composition on bee richness are greater on farms with low habitat diversity than on farms with high habitat diversity . Similarly, in Argentina, the importance of wildflower strips as pollinator sources for sunflower increased in the absence of large remnants of natural habitats nearby . In South Africa, the importance of weed richness for enhancing sunflower seed set increased with larger distances from natural areas . Throughout Europe, extensive programs aim to mitigate biodiversity loss on farmland through practices such as organic farming or wildflower strips, thereby offering a unique opportunity to understand interactions among these methods. A meta-analysis showed that these practices enhanced pollinator richness , but their effectiveness varied with the magnitude of increase in flowering plant cover resulting from the practices, farmland type, and landscape context . Because intensively managed croplands are generally devoid of flowering plants, pollinator-supporting practices in these landscapes result in the largest increase in floral resources and thus pollinator richness . On the other hand, conventionally managed grasslands generally contain more flowering plant species than arable fields,making it more difficult to enhance floral resources and pollinators . Finally, local effects were more positive in structurally simple landscapes than in cleared or complex landscapes, presumably because cleared landscapes lack sources of pollinator colonists and complex landscapes have less need of restoration. Recently, researchers have begun to explore the relative effectiveness of different pollinator-supporting practices. In Europe, flower strips were more effective than grass-sown or naturally regenerated strips . Globally, the effect of landscape composition and farm management was more important for improving bee richness than the effect of landscape configuration . Interestingly, conventional farms with high in-field habitat diversity maintained similar pollinator abundance as organic farms with low in-field habitat diversity, across the gradient of heterogeneity in surrounding land use. Thus, different combinations of local and landscape practices can result in similar outcomes in terms of promoting pollinator richness, providing alternative solutions suited to different agricultural settings. The importance of small-scale practices is likely greater for insects with short flight ranges foraging from a fixed nest, such as small- to medium-sized bees, which usually forage within an area of a few hundred meters and comprise the greatest fraction of bee species . Consistent with the idea that small-scale practices alone can have high impact, a study designed to separate the effects of local- versus landscape-scale habitat on pollination services delivered to blueberries found that the local scale had stronger positive effects . Indeed, farmers acting individually are more likely to improve the quality of their own fields and the immediate surroundings than to be able to manage complete landscapes for pollinators. Assuming a foraging range of 200 m from the nest for small bee species , diverse and high quality habitats need to be provided within 13 ha .Understanding the socioeconomic consequences of pollinator-supporting practices is essential to effectively enhancing wild pollinator richness in “real-world” landscapes . Farmers generally face implementation costs, such as those for planting hedgerows, and opportunity costs, such as those for setting aside natural habitats that could otherwise be cultivated .

This indicates that despite the drastic reduction in defense, some mechanisms have remained in domesticated types or have been recently introgressed by geneflow from wild populations. Identifying and amplifying these anti-herbivore defense mechanisms in modern cultivars, as well as introducing new mechanisms from wild relatives and landraces, could greatly improve productivity and profitability, while limiting the use of toxic agrochemicals. Like other crops, Lima bean has lost many of its mechanisms of defense against insect herbivores during the bottlenecks of domestication and modern breeding programs. This is illustrated by the finding that wild P. lunatus seedlings have greater chemical diversity than their domesticated relatives . Lima beans have been used as an experimental model in numerous studies of herbivoreinduced direct and indirect defenses . Previous studies have examined the role of cyanogenesis, volatile organic compounds, blueberry package and extrafloral nectaries as plant defense mechanisms against herbivores . Due to the high metabolic cost to the plant of producing these defensive compounds, P. lunatus makes tradeoffs between direct and indirect defense mechanisms .

The goal of the research presented In this dissertation Is to understand the domestication history, current variability, and breeding potential of the antiherbivore defense traits in Lima bean with a special emphasis on cultivars adapted to the Central Valley of California and cyanogenesis as a mechanism of defense against Lygus hesperus. Lima Beans as the Predominant Grain Legume in California Lima beans are one of five domesticated species in the genus Phaseolus. They were independently domesticated first in Central Mexico and again on the western slope of the Andes Mountains of Ecuador and northern Peru . Dating of starch grains on human dental remains, indicates that beans were domesticated in Northern Peru approximately 8000 years ago . Estimates for the date of domestication of Lima beans in Mexico are less certain but range from 2300-3400 years ago . The domesticated Andean gene pool is characterized by large flat seeds while the Mesoamerican gene pool has smaller round or flat seeds . Lima beans are multi-annual or semi-perennial, with crops typically needing 115- 135 days to reach maturity . They are adapted to a range of climates but are especially suited to warm and humid environments . Lima beans are grown in many regions of the world, including Africa, Asia, and Central and South America . In the United States, California is the primary growing region for mature dry Lima beans, where they are an important crop for the agricultural systems of the Central Valley.

As a nitrogen-fixing, highly vigorous rotation crop for tomatoes and other high-value crops, Lima beans provide an essential service of sustaining soil fertility and breaking pest and weed lifecycles . Additionally, Lima beans are very drought tolerant, making them ideal for the perennial water shortages experienced by California in recent decades . Two market classes – small white and large white – are grown in the state. Baby Limas are grown mostly in the region around Sutter and Colusa Counties. Large Limas, which need cooler nighttime temperatures, are grown mostly in Stanislaus County . Approximately 20,200 acres of Lima beans were grown in 2018, representing nearly half of the dry bean production in the state . Improved cultivars of Lima bean yield approximately 2,500-3,500 pounds per acre . Production of Lima bean in California is limited by its vulnerability to L. hesperus. Regular treatment in the field with pesticides, specifically pyrethroids, is the only known effective method of control . This practice is costly for a low-value crop like Lima beans and is unsuitable for organic production.Herbivory by the polyphagous, native Californian insect Lygus hesperus Knight , negatively affects the yields of several important crops, including alfalfa, strawberries, safflower, peaches, almonds, and dry beans . Current methods of controlling L. hesperus are costly, environmentally toxic, and only moderately effective . In sensitive crops like alfalfa and Lima bean, L. hesperuscan cause up to 70% yield loss as measured in sprayed versus unsprayed plots . There are typically four or five generations of L. hesperus each summer, with variability due to climate . The rate of development is dependent on temperature, but it takes on average 27 days from egg to reproductive adult at 20°C . Each generation develops from egg to adult with five nymphal instars going through incomplete metamorphosis .

All nymphs are flightless, but adults are highly mobile . L. hesperus are omnivorous but feed mostly on plant tissue . Their style of feeding is known as rupture feeding or “lacerate and flush.” Feeding starts with the insect probing the food tissue with its straw-like stylet, causing cells to rupture. Saliva secreted from the stylet has enzymes like polygalacturonase and -amylase, which further break down the tissue, creating a slurry that the insect can ingest through their stylet tube . L. hesperus feeding on Lima beans results in the abscission of flowers or young pods and consequently, yield loss. When feeding occurs on mature pods seed viability may be reduced and scarring can occur on seeds, thereby lowering market value . Adults spend about 20% of their time, and nymphs about 30% of their time, probing plants with their stylets. Actual ingestion represents only 3% of the probing time . The mechanical and chemical damage caused by this frequent probing and feeding behavior contributes to the heavy impact that L. hesperus have on crop yields.Understanding the mechanisms and inheritance patterns of L. hesperus tolerance or resistance in Lima bean will aid efforts to breed new varieties that require fewer pesticides and are suitable for organic production. To start, chapter 1 presents a comprehensive review of the literature on how domestication has affected the genetics of insect defense traits. Chapter 2 details the results of a genome-wide association study of cyanogenesis in Lima bean. The goal of this study was to explain how cyanogenesis has been affected by domestication in the Mesoamerican gene pool of Lima beans, with special consideration of the cyanogenic capacity of California cultivars. Chapter 3 will explore the variation and heritability of the tolerance or resistance to L. hesperus in cultivars of Lima bean adapted to the Central Valley of California. This will include a study of how cyanogenesis in Lima bean is affected by the presence of L. hesperus, specifically to determine the extent to which this trait is constitutive or induced. Analysis of the survival and reproduction of L. hesperus on varieties of Lima bean with variable expression of cyanogenesis will also be presented.The processes of cultivation and domestication have transformed wild species into crop plants that are an invaluable food source for the human population. These same processes have also made crop plants more vulnerable to damage by insect pests than their wild relatives . Globally, blueberry packaging insect herbivory accounts for an estimated 18-20% of yield loss during crop production . It is expected that these losses will increase if current trends in climate change continue . Recovering the defensive abilities of crop wild relatives in domesticated plants would result in reduced need for pesticides – which are harmful to human and environmental health – as well as an increase in global food security. Plant defenses against insect herbivores typically consist of complex suites of traits . These may include resistance traits like chemical deterrents, physical barriers, and reduced palatability, or tolerance traits like increased vigor and delayed phenology . Defensive traits can also involve attraction or resource benefits for beneficial organisms such as parasitoids and predators of insect herbivores .

Defense traits may be constitutively expressed and may also be induced to a higher level of expression by factors such as the presence of the target herbivore or volatile signals from a neighboring plant . Induction of a defense trait may occur immediately or slowly over time . The response can also be localized tothe area immediately surrounding the site of damage or be widespread throughout the plant . Several environmental factors can affect the expression of these traits including light intensity , the interspecific and intraspecific diversity of neighboring plants , photoperiod, temperature, and climate . Control of these traits typically involve the expression of multiple loci, which may be linked to other useful or unfavorable agronomic traits . The process of domestication involves selecting wild plants with desirable agronomic traits. The resulting crop plants typically exhibited some or all a suite of traits known as the domestication syndrome. This can include increased palatability, loss of dormancy, seed retention, increased seed size, and seed number . Selection intensity varied across crops and domestication events and there is serious academic debate about the duration and intentionality of this process . Identifying the genes that control agronomically important traits and understanding the way in which they have been affected by domestication is foundational to conserving, amplifying, and increasing their utility. This review will focus on the ways in which the process of domestication has altered the genes underlying plant defenses against insect herbivores. Prior reviews have focused on the mechanisms and physiology of plant defense against insect pests as well as the ways in which domestication has affected the interactions between crop plants, insect herbivores, and higher trophic levels . To my knowledge this is the first review on the subject of how domestication has affected plant and insect herbivore interactions with a specific focus on crop genetics.Domesticated gene pools have significantly less genetic diversity than wild gene pools . During the transition from wild plant to domesticated crop, diversity is lost during the actual domestication process as well as during cultivation, dispersal outside the center of origin, and later improvement through modern breeding . This loss of diversity within crop species has contributed to a loss of insect herbivore resistance traits . This is illustrated in maize through a comparison of teosinte, maize landraces from within the center of origin, maize landraces from outside the center of origin, and modern maize cultivars. Each stage of transition resulted in changes to the anti-herbivore defense strategies as well as changes in gene expression . A similar pattern can be seen in an analysis of the GsRbohA1 locus in soybean of which the A haplotype confers resistance against common cutworm . This allele was only present in 2.2% of modern cultivars compared with 23.5% of landraces and 95.6% of wild accessions . In the case of some anti-herbivore defense mechanisms, the selection against certain phenotypes had clear benefit to human consumers. For example, selections against genotypes which produce toxic compounds like cyanogenic glycosides, or distasteful physical defenses like trichomes, improved the safety and palatability of food . However, several important defense traits such as herbivore-induced volatile organic compounds cannot be perceived by casual observation and have only recently been revealed by careful scientific study . Despite the impossibility of direct selection for or against these traits prior to their discovery, several studies have identified differences in the expression between wild and domesticated crop plants. For example, in several studies of phytophagous insects and their parasitoids, parasitism was higher for hosts on domesticated rather than cultivated plants . In other studies, reduced predation or parasitism was attributed to the loss of chemical diversity or volatile signals . The loss or amplification of indirect anti-herbivore defense traits may be due to genetic drift, linkage, pleiotropic effects, or selection for alternate resource allocation. However, these were not intentionally selected against as their function has only recently been discovered . Breeding programs with little or no insecticide protection may help maintain insect defense traits compared to programs in which insecticides protect plants and mask susceptibility . Two early steps in the process of domestication were cultivation and storage, the intentional planting of future crop species and the saving of seeds between planting seasons . Both cultivation and storage created novel selection pressures on crop plants and their insect herbivores. Traits changed in frequency from the wild population when under the selection pressures of cultivation and storage . For example, in common bean, Phaseolus vulgaris, resistance to Mexican bean weevil Zabrotes subfasciatus is most likely due to a protein ofthe APA family, arcelin . Arcelin is only found in some accessions of wild Phaseolus vulgaris from Mesoamerica and is controlled by a single mendelian gene for which arcelin production alleles are dominant over alleles for its absence .

The relationship between diversity and the stability of communities and ecosystems is a fundamental question in theoretical and experimental ecology . Beginning in the 1990s, and sparked by clear experimental results showing that species richness decreased the temporal variance of ecosystem function , biodiversity research contributed to a broader discussion about how worldwide declines in biodiversity would affect ecosystem services on which humans rely . A major goal of biodiversity research has involved separating the relative importance of richness , composition , and abundance as drivers of temporal variance in ES. However, both the study designs and the analytical approaches used vary between experimental and observational studies. At smaller scales, the field of biodiversity-ecosystem function research has used controlled experiments and a well-developed body of mathematical theory to explore how species richness and composition affect temporal variance. In contrast, at larger scales, the field of biodiversity-ecosystem services research has been built mostly on correlative studies conducted in real-world systems —where ‘real-world’ means communities that are not directly manipulated—in which it is difficult to rigorously separate the causal roles of richness, composition and abundance .

Because species loss continues to occur at high rates worldwide , nursery grow bag it is critical to gain a better understanding of how species richness affects temporal variance of ES. This requires the development of novel analytical approaches that can separate the effects of richness, composition, and abundance without experimental manipulations, which are difficult if not impossible to conduct at landscape scales.An examination of ES in real-world systems is needed because controlled experiments that typify most biodiversity-ecosystem function research do not fully represent ecological reality ; specifically, these experiments do not mimic realistic species abundance distributions or species loss scenarios. First, a skewed or ‘hollow curve’ species abundance distribution, meaning that communities are composed of few common but many rare species, is ubiquitous in nature yet biodiversity-ecosystem function experiments have not mirrored this pattern and have instead used substitutive designs that equalize initial abundances among species . Although substitutive designs are arguably preferable for isolating the effects of species richness, they do make experimental communities less realistic and decrease the potential for one dominant species to provide the bulk of ecosystem function. Second, species are lost from communities non-randomly, with environmentally-sensitive and rare species being at greater risk of extirpation . In contrast, most biodiversity-ecosystem function experiments have assigned species to plots at random to avoid confounding species richness and species composition.

It is well-known that, because of this design, comparing high- and low-richness experimental plots reveals the effects of random species loss, which will under- or overestimate the effects of non-random species loss, depending on whether species with high or low contributions to function are lost first . However, biodiversity-ecosystem function researchers have countered that understanding the effects of random species loss is an important starting point, given that future patterns of species loss may be unpredictable . Throughout this paper, we use species loss to indicate local extinctions, because ES are delivered by local populations . Despite an awareness of these systematic differences between biodiversity-ecosystem function experiments and real-world ES, there is no consensus on whether species richness will contribute more or less to function when experimental results are scaled up to larger, more complex systems.Scaling up biodiversity-ecosystem function research is further complicated because analyzing observational data creates challenges not present in experiments. First, few if any real-world ecosystems allow researchers to independently assess services provided by each species in the community. This precludes the use of analytical approaches commonly used in bio-diversity ecosystem function research, especially analyses requiring single-species monocultures . Second, communities assemble and disassemble non-randomly with respect to species’ contributions to ES, making it difficult to separate the effect of richness from the effects of species identity. Third, the ubiquity of skewed species abundance distributions in nature makes it hard to separate the effect of abundance from species identity, if the same species are dominant across sites.

Because of these issues, no general analytical method exists for biodiversity-ecosystem services studies and, perhaps for this reason, no consensus exists on the importance of different components of biodiversity for ES . Here, we present a novel version of the Price equation that can analyze temporal variance of any ES, so long as the ES can be expressed as a sum of species-level contributions. Our work builds on the original Price equation from evolutionary biology, and its recent adaptations for biodiversity research . Fox provided the original framework for analyzing temporal variance with the Price equation, and here we extend it so that it can be used with observational data even if species composition is not nested between sites . Our version of the Price equation partitions between-site differences in temporal variance into three additive terms: variance in ES attributable to richness , composition , and context-dependence . Here, we use ‘abundance’ in place of‘context-dependence’ because in our data this term is determined by patterns of abundance fluctuation over time . The richness term establishes an expectation for how species loss and gain would affect ES if species were identical in the ES each provides. The abundance term quantifies the extent to which species present at both sites contribute more to temporal variance of ES at each site.We explore the temporal variance of pollination services using two large, multi-year datasets on pollination provided by bees. Using the new derivation of the Price equation described above, we ask: What is the relative importance of changes in species richness, composition, and abundance to the temporal variance of ecosystem services? Specifically, we compare the relative importance of richness and composition versus abundance.Field surveys. Our study systems consist of the wild bee pollinators of watermelon Matsum. & Nakai) and northern highbush blueberry  plants, both of which rely on insect pollination for successful fruit production. Over five years , we sampled wild bee communities at 10 commercial watermelon fields in central New Jersey and eastern Pennsylvania, USA. We also, over three years , sampled wild bee communities at 16 commercial blueberry fields in southern New Jersey. In a post-hoc analysis, plastic growing bag we confirmed that differences in our results between study systems were not due to length of sampling . Hereafter, we refer to these fields as sites. We ensured that all sites were at least 1 km apart, beyond the typical foraging radius of most bee species in our study . We did not include the honey bee in our data collection, primarily because in our system the honey bee is a managed species that is kept in hives and moved in and out of crop fields by bee-keepers and farmers. Thus, the temporal and spatial variation in honey bee abundance is driven by hive placement rather than ecological factors. In addition, honeybees are the property of bee-keepers and farmers, so we cannot collect them. Finally, honeybees are present at nearly all sites, so including them would likely increase the relative importance of ‘abundance’, making it conservative with respect to our findings to leave the honey bee out of the analysis.

Because watermelon is an annual species, farmers do not necessarily plant it in the same locations each year. To exclude potential effects of spatial variation on wild bee communities, we included watermelon sites in our analyses only if the maximum among-year distance between transects was ≤ 435 meters; this is within the typical foraging radius of all but the smallest bee species in our study . Pollination services. To measure bee richness and the pollination services delivered at each site on each date, we collected two forms of data: the number of individual wild bees visiting flowers, and the number of pollen grains deposited per flower visit. We then multiplied each species’ abundance by the mean pollen deposition of its morphological group to obtain that species’ contribution to pollen deposition. To measure bee abundance, we established a 50 m transect at each watermelon or blueberry site. We collected bees visiting flowers by net throughout the transect and then processed voucher specimens for species-level identification by taxonomists . At each site, data were collected on three days during each plant species’ peak bloom period, with three temporally-stratified 20-minute-long collections during the day; all data collection days were sunny, partly cloudy, or bright overcast with limited wind. Total collection effort was 135 days for watermelon and 144 days for blueberry. We measured the pollination efficiency per flower visit for different pollinator groups in field experiments. We offered a virgin flower to an individual bee foraging in the field, allowed the bee to visit the flower one time, and recorded the pollinator group of the bee . These pollen deposition experiments were conducted in three years for watermelon, and in two years for blueberry. Back in the laboratory, we use a compound microscope to count the number of conspecific pollen grains deposited during the single flower visit. To prepare slides, watermelon stigmas were softened in 10% KOH, and stained with 1% fuchsin. Blueberry stigmas were softened in 1 M NaOH, and then stained for 48h in 0.01% analine blue buffered in 1 M K3PO4. In the pollen deposition experiment, individuals were placed into morphologically similar groups which could be differentiated in the field . Due to the difficulty of collecting single-visit pollen deposition data, we did not collect these at all sites and in all years. However, we do know that that different morphological groups differed significantly in pollen deposition rates, while species within groups did not . Further details of all data collection methods, and site details, are available for both watermelon and blueberry . Because there is substantial variability within each morphological group’s distribution of pollen deposition rates, we conducted a sensitivity analysis to test whether choosing a single pollen deposition value and discarding the remaining variability affected our results. Instead of multiplying each bee species’ abundance by its morphological group’s mean pollination efficiency, for each individual bee, we randomly drew one pollination efficiency value from the correct morphological group’s distribution. We repeated this sensitivity analysis 1000 times, and found that the same results were obtained using either the ‘mean’ or ‘sensitivity’ versions of the analysis .In the watermelon system, we collected 3044 individual wild bees belonging to 59 species , 14 genera, and 5 families. Sites ranged in total temporal variance from 2.3 million to 51.1 million grains of pollen , in richness from 20 to 32 species , and in abundance from 140 to 435 individuals ; richness and abundance values are summed across years. In the blueberry system, we collected 1067 individual wild bees belonging to 36 species , 9 genera, and 4 families. Sites ranged in total temporal variance from 2.6 to 875,154 grains of pollen , in richness from 3 to 19 species and in abundance from 4 to 143 individuals . The range of richness values is similar to the range found in experiments that study various ecosystem services , although only two of our sites had species richness values similar to the lower end of richness manipulations . The rank-abundance distributions for each system are shown in Figure 1.The five Price equation terms are measures of directional effect size, where positive or negative values tell how strongly each term decreases or increases between-site differences in the total temporal variance of ES. For both systems, all richness-loss terms were negative and all richness-gain terms positive, with richness-loss greater in magnitude . This reflects the fact that total temporal variance of ES is generally higher at higher richness sites.All composition-loss terms were positive and all composition-gain terms negative, such that in all cases, composition terms partially cancelled their corresponding richness terms. This means that both the lost and gained species tended to have below-average contributions to total temporal variance of ES. Specifically, the positive composition-loss indicates that observed reductions in species richness resulted in less reduction in total temporal variance than would be the case if species losses had been random with respect to total temporal variance. Similarly, the negative composition-gain indicates that observed increases in species richness resulted in less increase in total temporal variance than would be the case if species gains had been random. In the watermelon system, composition-loss cancelled 86% of richness-loss and composition-gain cancelled 78% of richness-gain; in the blueberry system, these same value were 23% and 76%.

Five common conifer species occur on the northern range of the Archipelago: western hemlock , mountain hemlock , yellow-cedar, Sitka spruce , and shore pine . These coastal forests are simple in composition yet often complex in age and tree structure . Yellow-cedar occurs across a soil-drainage gradient from poorly drained bogs to well-drained soils on steeper slopes that often support more productive stands . This study occurs in the northern portion of the yellow-cedar population distribution and at the current latitudinal limits of forests affected by decline. We centered our investigation on protected lands in four inlets in the Alexander Archipelago on the outer coast of the West Chichag of-Yakobi Wilderness on Chichagof Island in the Tongass National Forest and Glacier Bay National Park and Preserve . Aerial surveys were conducted in 2010 and 2011 to assess the presence of affected forests and to identify the edge of yellow-cedar dieback that occurs south of GLBA on Chichagof Island. Aside from a brief history of small-scale gold mining that occurred in several areas on Chichagof Island between 1906 and 1942, plastic planters there is little evidence of human impact on these lands, making them ideal for studying ecological dynamics.

Drawing upon previous studies that estimated the time-since-death for five classes of standing dead yellow-cedar trees at various stages of deterioration , our plot selection consisted of sequential steps, in the field, to sample forests representative from a range of time-since-death. Not all yellow-cedar trees in a forest affected by mortality die at once; mortality is progressive in forests experiencing dieback . Highly resistant to decay, these trees remain standing for up to a century after their death . As a result, they offer the opportunity to date disturbance, approximately, and to create a long-term chronosequence. First, we stratified the study area coastline into visually distinguishable categories of ‘‘cedar decline status’’ by conducting boat surveys and assessing cedar decline status across 121.1 km of coastline in June 2011 and 2012. We traveled the coastline and made visual observations of live and dead yellow-cedar trees and their snag classes. We assigned cedar decline status to coastal forests at 100 m increments using a GPS Garmin 60 CSx . Next, using the ArcGIS 10.2 Geographic Information System software , we randomly generated plot locations in forests categorized during the coastline survey as follows: live, unaffected by mortality; recent mortality; mid-range mortality; and old mortality. Lastly, we controlled for basal area and key biophysical factors, including elevation and aspect via methods described.

Plots were restricted to elevations less than 150 m, excluding northeast facing plots, to sample from low-elevation plots representative of conditions where yellow-cedar decline commonly occurs at this latitude . Plots were randomly located between 0.1 and 0.5 km of the mean high tide to avoid sampling within the beach fringe area, and on slopes ,72% to limit risk of mass movement . We excluded plots with a total basal area ,35 m2 /ha to avoid sampling below the optimal niche of yellow cedar . This control was performed in the field by point sampling to estimate basal area using a prism with a basal area factor 2.5 . Plots dominated by the presence of a creek bed or other biophysical disturbance were eliminated from plot selection, due to the confounding influence of disturbance on the number of trees standing and species abundance. By restricting our sampling to these controls, our study was designed to examine the process of forest development post-decline in low-elevation coastal forests with plot conditions typical for yellow-cedar mortality excluding bog wetlands, where yellow-cedar may co-occur sparsely with shore pine. After controlling for biophysical factors, 20 plots were sampled in live forests and 10 plots in each of the affected cedar status categories for a total of 50 plots across the study area .Data were collected in fixed, circular nested plots to capture a wide range of tree diameters and in quadrats within each plot to account for spatial variability in understory vegetation.

Forty plots were established and measured during the 2011 field season and 10 plots during the 2012 field season, through the seasonal window of mid-June to mid-August. Nested circular plots were used to sample trees and saplings as follows: a 10.3 m fixed radius plot for trees 25.0 cm diameter at breast height , a 6.0 m fixed radius plot for saplings ,2.5 cm dbh and 1 m height, and trees 2.5–24.9 cm dbh. We counted live saplings of each species to analyze the population dynamics for individuals that survive to this size class. For each tree, we recorded species, dbh to the nearest 0.1 cm, height to the nearest 0.01 m, dead or live, and for dead trees snag classes I–V. Eight quadrats at each plot were utilized to record understory plants and tree seedling densities. To provide an additional longterm view of species changes, we recorded counts for smaller conifer seedlings , identifying western hemlock and mountain hemlock to genus, and other conifers to species. We noted presence/ absence of each conifer species 10–99 cm, but did not sample this size class for individual counts. We recorded maximum height and percentage cover of each plant species observed according to the Daubenmire method on a continuous scale . In unique cases where consistent identification to species was difficult Salisb.; Vaccinium ovalifolium Sm., and V. alaskaense Howell, we combined observations but noted both species presence for total richness across the study area. Blueberries, V. ovalifolium and V. alaskaense, are similar in appearance and often synonymized . Mosses and liverworts were recorded together as bryophytes within the quadrat. Sedges were recorded together but distinguished from true grasses . We used hemispherical photography to assess canopy cover at each plot. Photographing from plot center at dbh camera height, we captured imagery in relatively uniform, overcast skies and consistently avoided any mid-day sun conditions . To prevent diminished sharpness associated with consumer-grade cameras , we used a Sigma 4.5mm fish-eye lens on a professional grade Canon 7D camera . Full-view images were processed using Gap Light Analyzer to yield percentages of canopy openness per plot as a proxy for light in understory analyses .Clustering plots by cedar decline status.—To rigorously account for the timing of mortality relative to the coarse visual cedar decline status categorizations made by boat, we performed kmeans clustering analyses on the yellow-cedar population observed across the chronosequence by partitioning 50 plots into those affected by mortality and live ‘‘controls’’ for subsequent stages of analysis. Using observations of dead and live yellow-cedar trees at each of the 50 plots , we stratified the plots into two groups for unaffected and affected forests. We then performed a k-means clustering analysis with the categorical snag classifications observed at the resulting 30 plots affected by mortality, plastic plant nursery pot assigning the a priori k ¼ 3 for three affected status categories sampled: recent mortality, midrange mortality, and old mortality. We restricted this analysis to yellow-cedar trees .10 cm dbh because the methods of dating time-since-death for yellow-cedar trees rely upon standing, larger trees . We analyzed the cluster stability by computing the Jaccard coefficient to measure similarity between resulting clusters, assessed by the bootstrap distribution of the Jaccard coefficient for each cluster compared with the most similar cluster in the bootstrapped datasets .

Post hoc Fisher’s exact tests further clarified differences in the numbers of observed class I, II, and III snags between recent and mid-range mortality clusters ; observed class II, III, and IV snags between recent and mid-range mortality clusters , and between mid-range and old mortality clusters ; and observed class III, IV, and V snags between mid-range and old mortality clusters . These analyses were performed in R using the GCLUS and FPC packages. This post-field methodology for plot stratification enabled us to refine the visual cedar decline status assigned in the boat surveys by clustering according to the observed populations of live yellow-cedar trees from the plot data. Stand structure and regeneration.—We calculated the importance value for live conifers in the overstory as the sum of relative density, frequency, and basal area per species to characterize the stand structure and conifer composition within each cedar decline status resulting from clustering analyses, and to make comparisons across the chronosequence of cedar decline status. For each species in three size classes , we computed the following variables: density , frequency , and dominance , and with the relative values of these three parameters, the importance value was calculated as IV ¼ DR þ FR þ DoR . Thus, the cumulative value for all tree species per size class in each cedar decline status was 300%. In assessing regeneration, we focused analysis on seedling counts and saplings to consider established plants. We used Krukal–Wallis tests and performed permutation tests on the measure of central tendency to examine differences in mean seedling and sapling abundance across the four cedar decline status categories. Using presence/absence sapling data, we calculated the probabilities of finding each individual conifer species in the sapling life stage in each cedar decline status and generated binomial confidence intervals to estimate uncertainty using the Wilson score interval. We used a two-part modeling approach to determine the probability of species’ occurrence in cedar decline status and to test for significant effects of cedar decline status on each species’ abundance in the sapling stage. This method was selected to account for over dispersion in zeros in the individual abundance data for the conifer species in the sapling life stage . In the first step, the data were considered as zeros versus non-zeros and a binomial model was used to model the probability of observing a zerovalue; in the second step, non-zero observations were modeled with a zero-altered Poisson model . Canopy openness and cedar decline status were included in the models as explanatory variables to predict species presence/absence and sapling abundance. Best models were selected based upon AIC values. These analyses were performed in R using the PSCL, MHURDLE, and BINOM packages. We determined the IV for saplings in each cedar decline status on the basis of relative density and relative frequency , such that the IV of all species would sum 200%. To compare the persistence of saplings to treelets in the early stages of stand development, we calculated the ratio of saplings to live treelets per hectare at each plot and tested for significant differences between live ; recent mortality and live ; mid-range mortality using Wilcoxon rank sum tests. Probabilities calculated for species occurrence in the size class 10–99 cm in each cedar status were used for comparison with seedling and sapling results to assess trends in survival.The changes observed across the chronosequence provide strong evidence that this species dieback associated with climate change can result in a temporally dynamic forest community distinguished by the diminished importance of yellow-cedar, an increase in graminoid abundance in the early stages of stand development, and a significant increase in shrub abundance and volume over time. Tree mortality timing and intensity, as characterized by our stratified sampling of cedar decline status, played an important role in determining the understory community composition and overstory processes of stand re-initiation and development. Our results highlight the ways in which widespread mortality of one species can create opportunities for other species and underscores the importance of considering long-term temporal variation when evaluating the effects of a species dieback associated with climate change. Methods for predicting future changes in species distributions, such as the climate envelope approach, rely upon statistical correlations between existing species distributions and environmental variables to define a species’ tolerance; however, a number of critiques point to many factors other than climate that play an important role in predicting the dynamics of species’ distributions . Given the different ecological traits among species, climate change will probably not cause entire plant communities to shift en masse to favorable habitat . Although rapid climatic change or extreme climatic events can alter community composition , a more likely scenario is that new assemblages will appear . As vulnerable species drop out of existing ecosystems, resident species will become more competitive and new species may arrive through migrations . Individual species traits may also help explain the process of forest development in forests affected by widespread mortality, as the most abundant species may be those with traits that make them well-adapted to changing biotic and abiotic conditions .

California is an agricultural hot-spot of the world, producing large quantities of field crops, fruits, nuts and vegetables. In 2019 the total value of principal crops in California was over $33 billion, the second-most productive state was less than half that amount . There are estimated to be between 1,600 to 2,000 species of bees in California , of which 46 bee species within 17 genera are found commonly in Californian cities . Bees are indispensable drivers of ecosystem pollination, aiding greatly to plant reproductive success and biodiversity , the foundation of an enormous diversity of other organisms. Effective bee pollination across a variety of landscape types helps to ensure self-sustainable cities and human communities . This research focuses on the connection between plants and the attracted foraging bees. This study connects the floral landscape to pollinator foraging events taking place on it. For example, a landscape’s plant community composition is known to be a main predicting factor in the presence or absence of bees .

Many studies focus on bee foraging habitats and have tried to quantify the attractiveness of plants to bees . While plant list short-comings are part of the problem with evaluating bee habitats , french flower bucket understanding how spatial implications play out across a landscape is essential to enhance understanding of patch dynamics and the potential effects of fragmentation. As noted above, in Chapter 1 we determined that actual bee foraging preferences varied greatly from published expected foraging associations. This research builds on Chapter 1’s findings, by examining if spatial and temporal habitat usage was limited by a poor understanding of bee forage plants or not. This research study compares the smaller WHR plant selection with the full plant selection which bees used for forage in Chapter 1. By taking this approach, this research examines spatial habitat utilization and infers potential implications . The study examines whether the differences in plant palette usage made a large difference or not in utilized habitat.Habitat fragmentation, degradation and destruction are cited as the main reasons for declines in California native bee populations .

Drawing from the theories of island biogeography and metapopulation dynamics , fragmentation is defined as suitable habitat patches being too far apart to support sustainable bee populations. On an individual level fragmentation could lead to isolation, or the inability to disperse to another habitat patch for requisite resources, for example, lack of flowers . Spatial isolation of habitat patches has been found to decrease diversity of bees in urban landscapes . Even in naturalistic habitat fragments, there have been shifts of bee abundance and richness due to isolation . Overall, anthropogenic disturbances have significant negative effects on wild bees . However, in areas with less than 50% impervious surface pollinators still seem to provide sufficient pollination services to wild vegetation and crops . Currently bee life history literature does not concur on which scale of habitat fragmentation has the largest impact. For example, some studies concentrate at the entire world scale the North American continent scale , the entire United States country scale , and the smallest studies are said to be at the microsite, consisting of several point locations, several miles across , additionally, Cane et al. compared bee ecological characteristics such as dietary breadth.

Most of the above mentioned research focused more on European honey bees, though studied less often native bees were included in some studies. There are landscape elements which are completely inhospitable to bees and can be defined as habitat destruction, for example: paved surfaces and the footprint area buildings use . Habitat degradation to bees consists of detrimental changes of a site. For example, a dramatic shift in vegetation cover of a formerly naturalistic site will have effects on the bees which utilized the former floral resources. Bees’ responses to such shifts will vary , as their foraging habits, obligate versus generalist, are similar to butterflies in California . In terms of foraging some bees have benefitted from predominantly human shaped and dominated landscapes , while other bees decline . Habitat gaps may also occur temporally, depending on plant phenological patterns, leading to patch isolation or connectivity . Bee foraging preferences, whether polylectic or oligolectic , can also be an origin of habitat fragmentation if a landscape is not suitable. For bees, if sufficient floral resources are not available, that represents a non-foraging area, possibly a population sink. This phenomenon may lead to ecological filtering, reducing bee diversity . Current bee foraging plant lists have limitations in their ability to predict a bee genera’s presence or absence . Nevertheless, measuring bee-to-flower associations is a starting point to understanding the potential that there is habitat fragmentation for bees. Identification of the most basic levels where and when habitat fragmentation occurs is essential to determine before conservation remedies can be prescribed. It is essential to investigate these spatial problems with geographic tools to aid in bee conservation using empirical relationships with which bees experience their world.Bees vary widely in their foraging distance ability from a nest . Bee body size has been shown to be an indicator of forage diameter radii . Based on published body size data , bees may forage up to 2 mi maximum for only European honey bees, 1 mi for large bodied bees such as Bombus and Xylocopa, mid-sized bees are most common and average 0.25 mi , and the smallest bees are estimated at only 0.11 mi from their nests . Bee foraging radii is traditionally measured by the distance between a bee’s nest and its foraging range . However, determining individual nesting locations for solitary native bees is extremely difficult and not attainable or reliable based on current bee research studies , often taking a long time and often not gleaning results quickly enough for this research spatial scale and quantity of data . Given the intractability between locating nests of individual bees observed foraging in gardens, a general assumption is made in this study that foraging instances are relatively closely linked in proximity to nest locations, and therefore the foraging data points are used as a proxy for nesting locations. These foraging radii are for female bees, bucket flower which create and care for nests, rather than males, which utilize the landscape differently . Importantly, maximum foraging ranges represent an extreme limit on travel, as most individual bees will not travel as far as the maximum . In traditional meta population literature, differences between home range movement versus individual dispersal have been studied with more charismatic animals , but these movement dynamics have not been studied thoroughly in relation to bees. Among the limited extant bee dispersal research studies, results align with previously cited research regarding bee body size, degree of host plant specialization and bee sociality .The human landscape’s horticultural gardens can be florally diverse, contributing to an array of potential habitat types for bees . Furthermore, bee foraging preferences are not well understood, but are crucial to mitigate pollinator population declines . Thus, the role that horticultural garden conditions can contribute to increasing bee population numbers needs to be identified. Conversely, horticultural conditions which provide little to no benefit to bees should also be identified and avoided or discouraged. Studies conducted thus far have indicated a variety of responses of bees to managed landscapes, including both increased bee richness in some anthropogenic sites or some have shown decreased bee diversity in anthropogenic sites . However, overall, many land use activities are known to be detrimental to bee communities . Conversely, habitat simplification is known to have a negative effect on bee populations . Furthermore, plant community composition across a spatial gradient has been reported to have an effect on plant-pollinator resilience as a whole .

Therefore, it is important to assess both composition and configuration ofanthropogenic landscapes to determine why pollination ecosystem services occur in greater or lesser frequency across the landscape . Floral abundance, richness and spatial distribution have been noted to affect native bee communities , but the effect of these variables needs to be explored in more detail, and across a larger variety of garden types. Presence of weeds seems to enhance bee presence as well . Overall, bees respond differently to anthropogenic conditions and it is essential to study the variety of responses to maximize conservation efforts . Overall, current habitat remedies involve providing adequate floral resources and increasing floral diversity . Reconciliation ecology, a conservation science technique used in human dominated habitats , has been promising for other organisms and is highly applicable for bees.In recent years, scientists have used the term “Anthropocene” to describe the current geological epoch which by definition states how far humans have influenced Earth’s global biogeochemistry. Similarly, at the landscape scale, humans have dominated and changed Earth’s terrestrial ecosystems dramatically, leading to the term “anthroscape” . Notable in recent times, our infrastructure often excludes wildlife and represents an ecological sink or void. For example, paved roads offer no value to bees. Buildings and their footprints aid in reducing viable bee habitat. Notably, some horticultural planting schemes may represent opportunities to bees. Waterways, ponds, pools represent sinks to nearly all bees, the exception being Apis melifera which has low tolerance for critical water content and need fresh water to survive whereas native California bees have very high tolerance for critical water content and do not need fresh water bodies regardless of being in urban or rural ecosystems . The degree to which bees living in human-dominated areas act as source habitats to adjacent landscapes should be explored further.This research study aims to pinpoint when and where various bee genera experience habitat patch dynamics versus habitat fragmentation . Though bee and flower phenology are discussed at length in the literature, it has not been formally explored from an explicitly spatial perspective at the landscape scale. As such, this is the first study to document bee habitat spatial and temporal dynamics providing important novel insight for future bee conservation efforts or further studies in bee habitat fragmentation. The main research questions covered in this paper include: What constitutes a gap in native bee pollinator habitat? How do bees’ annual spatial patterns of potential versus realized habitat function over a year? How can bee foraging maps be used to conduct conservation gap analysis at a local scale? How do partial and full WHR plant palettes compare in terms of habitat modeling for bees? and, What are the implications of this research for bee habitats?To help solve these complicated topics, the wildlife-habitat relationship models developed for bees in Chapter 1 are used here to spatially estimate and measure gaps in bee genera pollination networks. The spatial analyses presented in this study shed light on the degree of real-world bee habitat fragmentation. Monthly observed bee georeferenced data which were compiled in Chapter 1, are here analyzed via GIS maps. Seasonality of both bees and flowers were integrated in the mapping model analysis in an effort to identify where and when bee potential habitat gaps and potential fragmentation might occur The spatial distribution patterns of suitable, insufficient, or unsuitable habitat for bees are still poorly understood, yet essential to understand for native bee conservation purposes. This study investigates if bee foraging maps can be used to conduct both spatial and temporal habitat gap analysis.The UC Davis Arboretum and Public Garden , located in California’s Central Valley, is a unique environment to study bee patch dynamics and potential habitat fragmentation. The curated plant collection is fully mapped and contains 35 gardens along the linear Arboretum landscape . Each themed garden has a distinct geographically defined border, and plants are identified/labeled to the plant species, subspecies, or cultivar level. Arboretum maps are spatially accurate within two meters .Computer geographic information systems technology, using ArcGIS version 10.7.1 , was employed to investigate and calculate where and when habitat gaps occur for bees in the anthroscape. The theoretical framework was based on wildlife-habitat relationships conservation science modeling . In Chapter 1, monthly WHR foraging models were developed for each bee genus observed in the Arboretum. This chapter applies the predictive foraging models spatially, using the Arboretum’s geodatabase of mapped plants. Using this approach, a GIS predictive bee foraging model was developed and tested on-site with fieldwork to verify and quantify differences .

As such, more work can be done on how disturbances alter seed microbiome assembly processes and outcomes.The genus Vitivirus was created in 1997 for the classification of type member grapevine virus A , a plant virus discovered in grapevine with a filamentous flexuous particle differing from trichoviruses in its genomic arrangement. Vitiviruses have a single-stranded RNA genome encoding five genes: replicase , movement protein, coat protein , nucleic-acid-binding protein and a 20 kDa protein of unknown function. In the 2018 International Committee of Virus Taxonomy Master Species List , nine species of vitivirus infecting grapevine are recognized: Grapevine virus A, Grapevine virus B, Grapevine virus D, Grapevine virus E,Grapevine virus F, Grapevine virus G, Grapevine virus H, Grapevine virus I and Grapevine virus J. Since 2019, two more proposed vitiviruses were discovered in grapevine. Grapevine virus L was initially identified in RNAseq data and later detected in multiple plants in Croatia, New Zealand and the United States. Grapevine virus M was also discovered by high throughput sequencing in an American hybrid grapevine. Three different vitiviruses have been associated with the etiology of rugose wood disease in grapevine, a disease with world-wide distribution.

GVA is associated with stem grooving on the variety Kober 5BB, grapevine virus B was identified as the causal agent of corky bark in the variety LN33, procona florida container and grapevine virus D was implicated in growth reduction in the rootstock Freedom. Additionally, these vitiviruses are frequently detected in coinfection with grapevine leafroll viruses, resulting in synergistic interactions that can lead to lethal effects in several scion and rootstock combinations. The potential pathogenic role of the remaining grapevine vitiviruses, including proposed members, is still unknown. Reliable diagnostic methods are critical in determining the viral infection status of a grapevine. Multiple tests are available for the detection of vitiviruses, including biological indexing, real-time or end-point reverse transcription PCR and HTS. Biological indicators do not show symptoms for all vitiviruses infecting grape, and RT-PCR assays can fail to detect vitivirus variants containing nucleotide differences at critical primer binding locations. HTS is the most effective means of detecting all vitiviruses but its high cost at large scale limits its use as a screening tool. HTS data is helpful to inform and update RT-PCR primer design as new virus strains are continually being characterized. In this study, a universal end-point RT-PCR assay involving degenerate primers with the capacity of detecting all the known grapevine vitiviruses was developed.

To validate the new assay, eleven grapevines each infected with one of the vitiviruses were tested. Moreover, a field survey was conducted of known vitivirus-infected grapevines. Following the first reports of vitiviruses in grapevine, several vitiviruses have been discovered in other hosts; consequently, we investigated if the universal assay can detect these vitiviruses.A universal assay able to detect all known grapevine vitiviruses and potentially other members of the genus Vitivirus was developed here based on sequence data available in GenBank. The presence of highly conserved motifs in the REP protein allowed the design of end-point RT-PCR primers, providing an alternative assay to reduce the work associated with the diagnosis of vitiviruses. The extensive sequence divergence existing among grapevine vitiviruses, observed at the nucleotide and aa levels, makes it difficult to design a test with broad-range detection. RT-PCR with degenerate primers is a simple strategy that is frequently used for the specific and simultaneous detection of multiple viruses. Assays involving degenerate primers targeting grapevine vitiviruses have been described before, however, these studies were conducted in the pre-HTS era, when fewer vitiviruses were known and sequence data was limited.

Although Vitis spp. is recognized as the main host associated with the genus Vitivirus, vitiviruses have been identified in other perennial hosts, the majority of which are woody plants. For example, vitiviruses have been reported in blackberry, mint, agave and recently in blueberry. The universal assay successfully detected MV-2 in mint, however failed to detect BGMaV in blueberry. Additional investigation revealed a variation in motif A of BGMaV , and a similar scenario was observed in AVV. Based on our PCR results we predict that the universal assay will miss AVV during diagnosis, though, the rest of the known vitiviruses do not display any a discrepancy in motifs A or B and they should be detected by the assay. The family Betaflexiviridae comprises twelve different genera , including Vitivirus, Trichovirus and Foveavirus. After in-silico and in-vitro analyses of trichoviruses and foveaviruses, we did not find evidence for cross-reaction by the universal assay. A single test for all known grapevine vitiviruses can be a useful tool for improving efficiency and reducing costs of large-scale surveys. Potentially, this generic assay may detect novel Vitivirus species in grapevine and other hosts given its unbiased nature. Similar assays have been developed for carlaviruses, nepoviruses and different members of the family Betaflexiviridae. Grapevine is clonally propagated, consequently, to prevent the spread of vitiviruses, it is critical to use virus tested material. The assay developed here will be made available to diagnostic labs and will facilitate the production of certified virus-tested propagation material and the effective control of vitiviruses.Over the last few decades, the field of nutrition has grown and evolved. Although we continue to define the critical roles that nutrients play as fuel sources, enzyme cofactors, signaling molecules, and vital infrastructure for our bodies, the cutting edge of nutrition research is pushing beyond simply meeting our bodies’ basic needs. Indeed, as the population is living longer, an emerging focus for nutrition has been on obtaining and maintaining optimal health over the life course. On 10 October, 2022, the Council for Responsible Nutrition held their annual Science in Session conference entitled Optimizing Health through Nutrition – Opportunities and Challenges. The audience consisted of scientists and executives from dietary supplement and functional food companies as well as nutrition graduate student awardees of a CRN and ASN Foundation educational scholarship to attend the symposium. CRN is a trade association representing dietary supplement and functional food companies. The goals for this meeting were to propose a definition for optimal nutrition and identify strategies and tools for evaluating optimal health and nutrition outcomes while highlighting the gaps in this emerging space. Now more than ever in history, our population’s health has emerged as a global priority. Currently, 6 in 10 adults in the United States have a chronic disease, and 4 in 10 have 2 or more. In <10 y, the number of older adults is projected to increase by ~18 million. This means that by 2030, 1 in 5 Americans is projected to be 65 y old. As the major risk factor for many chronic illnesses is age, it is anticipated that the rates of all age-related diseases, especially chronic diseases, will skyrocket, potentially overwhelming the health care system. We need to enable the health care system—and the population—to be more proactive rather than reactive toward health outcomes. There is a critical need to help find solutions to optimize health across the lifespan to support living better longer, i.e., health span. Ensuring optimal nutrition is a significant and easily modifiable variable in the solution for maintaining and improving health span. We need to advance concepts beyond essential health and consider meeting the nutritional needs for optimal health. Although the nutrition science community is moving toward the vision of nutrition to support optimal health, many challenges and gaps still exist, but there are also recent advances and exciting opportunities.

The goal of the CRN “Science in Session” workshop was to discuss these challenges, gaps, and opportunities in order to advance the concept of nutrition for optimal health. This review summaries these findings and discussions.The DRIs for individual nutrients, procona London container including the Estimated Average Requirement and the RDA, are life stage- and sex specific recommendations for Americans and Canadians. These reference intakes were established in the 1990s by the Food and Nutrition Board of the National Academies of Sciences, Engineering, and Medicine to prevent deficiency disease and to reduce the risk of chronic diseases. However, incorporating chronic disease endpoints has been extremely challenging, primarily because data are largely lacking. Thus, the current DRIs, including the RDAs that are aimed to cover the nutrient needs of 98% of the population, do not account for the amount of a nutrient that one needs in order to achieve and maintain ‘optimal’ health.The science of resilience is not a new concept—this scientific concept was documented in the literature as early as the 1800s; the terminology entered the biomedical sciences in the mid- 1900s and emerged in the early 2000s as a concept to be interconnected in multiple health domains. The questions dominating its broad use and applicability tend to focus on how to define resilience. In 2019, the Trans-NIH Resilience Working Group was formed with a goal to develop an NIH-wide definition of resilience and to achieve consistency and harmony on the design and reporting of resilience research studies. In 1993, an introductory manuscript to a special issue published on the science of resilience included a quote stating, “resilience is at risk for being viewed as a popularized trend that has not been verified through research and is in danger of losing credibility within the scientific community”. The authors of the manuscript also warned against definitional diversity with respect to measures of resilience and urged researchers to clearly operationalize the defi- nition of resilience in all research reports. Remarkably, this call to action served as a primary aim of the Trans-NIH Resilience Working Group when it was organized >25 y after the 1993 special issue on resilience. One of the first activities of the Trans-NIH Resilience Working Group was to host a workshop, in March 2020, which led to the development of a definition of resilience and a conceptual infographic. The definition was intended to be applicable and useful across multiple domains, and it states that resilience encompasses “A system’s capacity to resist, recover, grow, or adapt in response to a challenge or stressor” . A system can represent different domains, levels, and/or processes. Over time, a system’s response to a challenge might show varied degrees of reactions that likely fluctuate in response to the severity of the challenge, the length of time exposed to the challenge, and/or innate/intrinsic factors. To show applicability of the definition in resilience research studies, the Resilience Research Design Tool was later developed to help improve consistency in resilience research reports and to facilitate harmony with respect to measures of resilience outcomes. One of the goals of the resilience framework is to reframe the way we ask research questions, particularly about nutritional interventions like dietary supplements, so that we can better understand health outcomes that are not based solely on disease end points. Going forward, as researchers across various scientific domains and sectors come closer to a unified definition of resilience and perhaps agree to the use of a standard checklist for designing and reporting on resilience studies, there is greater opportunity to harmonize the science and develop more empirical evidence of resilience outcomes.Optimizing performance also includes building resilience in order to enhance the ability to perform tasks and ensuring resilience in order to prevent illness, injury, and disease. Within the US Department of Defense, researchers are able to study different models of physical and psychological stress and the application of different nutritional interventions with Service Members throughout their careers. Various models of stress are introduced, including initial military training , advanced military training courses , service academies , and extreme environments , along with examples of various interventions and outcome measures collected to date. The importance of nutrition on readiness and resilience was identified in military populations more than a decade ago and continues to be of interest. Two specific examples are provided to further explore nutrition interventions aimed at optimizing performance in the Department of Defense. The first, a completed double-blind, randomized, placebo-controlled trial, used a calcium and vitamin D fortified food product to optimize bone health during initial military training of Marine Corps recruits. Using a supplement or food intervention for calcium and vitamin D, participants received 2000-mg calcium and 1000-IU vitamin D per day. The primary outcomes of the study showed that bone markers and vitamin D status improve, but the supplementation did not affect skeletal parameters.

In contrast, the floral odors that attract moth pollinators have been more extensively researched. In this study we determined that CMV infection induced changes in olfactory cues emitted by Arabidopsis thaliana and tomato plants in ways that could be perceived by the bumblebee Bombus terrestris, and confirmed in tomato that this was associated with quantitative and qualitative changes in the blend of plant-emitted volatile organic compounds . We also elucidated a role for the host microRNA pathway in regulating the emission of bee-perceivable olfactory cues. Our data indicated that bumblebees possess an innate preference for olfactory signals emitted by CMV-infected tomato plants and we mathematically modeled what the possible wider implications of this might be if a similar preference occurred in wild host plants under natural conditions.In ‘free-choice’ assays, bumblebees encountered flight arenas containing ten tomato plants concealed within towers designed to allow odors to diffuse out but prevent the bees from seeing or touching the plants . Cups that were placed on top of towers hiding plants of both treatment groups offered bumblebees the identical ‘incentive’ of a 30% sucrose solution. Nonetheless, plastic planter pot when presented with mock-inoculated and CMV-infected tomato plants, bumblebees preferred to visit the towers that were hiding infected plants .

Bumblebees showed similar preferences for flowering and non-flowering CMV-infected plants, indicating that leaves were the main source of attractive volatiles . Bumblebees also displayed a preference for CMV-infected tomato plants over plants infected with CMVΔ2b, a viral mutant lacking the gene for the 2b VSR , a factor that also influences CMV-plant-aphid interactions.The results obtained in free-choice assays with tomato plants infected with CMVΔ2b suggested that the 2b protein, which is a VSR, may be exerting effects on the metabolism of plant volatiles by interfering with host small RNA pathways. The model plant Arabidopsis is the best higher plant system to use to investigate the effects of small RNA pathways. However, whilst Arabidopsis plants emit potentially pollinator-influencing volatiles, this species is not bee-pollinated. Consistent with this, bumblebees showed no significant difference in preference for volatiles emitted by CMV-infected versus mock-inoculated Arabidopsis plants in free-choice assays . An alternative approach to investigate the ability of bees to recognise differences in olfactory or other stimuli is to set up a differential conditioning or ‘learning curve’ assay. A differential conditioning assay can reveal whether bees can perceive cues that would not normally induce any behavioural responses and that could not be studied in free-choice assays.

In our differential conditioning assays, cups on towers offered bumblebees either a 30% sucrose solution ‘reward’ for choosing one treatment group or a ‘punishment’ for choosing the other group. Bumblebees cannot distinguish quinine from sucrose except by taste. Thus, increasing frequency of visits to sucrose-offering towers over the course of an experiment indicated that bees have learned to use plant odor as a cue to identify and avoid drinking from cups placed on towers offering quinine solutions. In these assays, a steep learning curve shows that bumblebees can easily distinguish between two treatment groups, and indicates that the volatile blends are likely to be qualitatively and/or quantitatively very distinct, whereas less steep curves indicate that differences between blends are less marked, and that bees find it more difficult to learn to distinguish between them based on odor. An illustration of the power of this approach is shown in Fig 2 . Although bumblebees displayed an innate preference for volatiles emitted by CMV-infected tomato plants in free choice assays , they could be trained by differential conditioning to overcome their innate preference and instead preferentially visit mock-inoculated tomato plants and avoid CMV-infected plants . Although we had observed that bumblebees had no innate preference for, or aversion to, volatiles emitted by Arabidopsis plants , differential conditioning assays revealed that the insects could recognize differences between volatiles emitted by Arabidopsis plants that had been mock-inoculated and by plants that were infected with CMV . Bumblebees could also distinguish between CMV-infected and CMVΔ2b-infected Arabidopsis plants .

Hence, although they exhibit no innate behavioural response to the volatile blends emitted by Arabidopsis plants, differential conditioning assays showed that bumblebees could perceive differences in volatiles emitted by these plants. This meant that differential conditioning assays could permit further dissection of the mechanisms underlying CMV-induced changes in volatile emission using Arabidopsis as a model system. Bumblebees could learn to differentiate transgenic plants constitutively expressing the 2b VSR from non-transgenic plants and from control-transgenic plants that were expressing an untranslatable 2b transcript . However, the insects displayed less ability to learn to distinguish mock-inoculated from CMVΔ2b-infected plants . Comparison of the learning curves in Fig 3A versus Fig 3E by logistic regression indicated that bumblebees were better at distinguishing mock-inoculated plants from CMV-infected plants than from CMVΔ2b-infected plants = 40.17, p < 0.0001. Bees could not be trained to differentiate non-transgenic plants from control-transgenic plants expressing a non-translatable 2b transcript . The results with CMVΔ2b suggested that the 2b VSR plays an important role in altering the emission of bee-perceivable olfactory cues emitted by tomato and Arabidopsis plants . However, CMVΔ2b accumulates to lower levels in plants than wild-type CMV and in previous work it was found that viral titer, as well as the presence of the 2b protein, plays a role in modification of the interactions of Arabidopsis with aphids. Hence, it was conceivable that differences in virus titer might affect the emission of bee-perceivable volatiles by plants infected by CMV or CMVΔ2b and explain why the bees found it difficult to distinguish CMVΔ2b-infected plants from mock-inoculated plants. However, it is known that CMVΔ2b accumulates to levels comparable to those of wild type CMV in Arabidopsis plants carrying mutations in the genes encoding the Dicer-like endoribonucleases DCL2 and DCL4, which are important factors in antiviral silencing. Therefore, we examined the ability of bumblebees to learn to distinguish between volatile blends emitted by CMVΔ2b-infected and mock-inoculated dcl2/4 double mutant plants . The resulting learning curve was not significantly different from that obtained using wild-type plants that had been mock inoculated or infected with CMVΔ2b = 0.66, p = 0.42, indicating that an increase in CMVΔ2b titer did not enhance bee learning. Although we cannot rule out a role for other CMV gene products, the results indicate that the 2b VSR is the most significant viral factor conditioning changes in the emission of bee-perceivable volatiles.One of the host molecules that interact with the 2b VSR is the Argonaute 1 ‘slicer’ protein. AGO1 is required for silencing directed both by short-interfering RNAs and by miRNAs, which are generated by a specific host endoribonuclease from miRNA precursor transcripts encoded by nuclear genes. In differential conditioning assays, bumblebees were able to learn to distinguish between volatiles emitted by wild-type plants versus those emitted by ago1 mutant plants and those emitted by dcl1 mutant Arabidopsis plants . However, bumblebees showed little or no ability to learn to distinguish between volatile blends emitted by ago1 and dcl1 mutant plants, 30 litre plant pots indicating that the volatile blends emitted by plants of these two mutant lines were very similar . Thus, the miRNA-directed silencing pathway regulates the emission of bee-perceivable volatile compounds. Double mutant dcl2/4 plants are unable to generate CMV-derived short-interfering RNAs but are not affected in miRNA biogenesis. In CMV-infected dcl2/4 plants a higher proportion of the 2b protein is available to bind AGO1 and inhibit its miRNA-directed activity, which is likely to enhance virus-induced changes in emission of bee-perceivable volatiles. In line with this, bumblebees were able to learn to distinguish between volatiles emitted by CMV-infected wild-type and dcl2/4 double mutant Arabidopsis plants . As an additional control we showed that bumblebees could not learn to distinguish between volatiles emitted by mock-inoculated plants covered by towers offering sucrose rewards or quinine punishments .

The responses of bumblebees to CMV-infected tomato plants that were hidden from the insects indicated that changes in the emission of volatiles were affecting bee behavior and were responsible for the innate preference of these insects for CMV-infected plants . To confirm that CMV infection caused changes in the emission of VOCs, tomato plant headspace volatiles were collected and analysed by gas chromatography coupled to mass spectrometry . VOCs were collected from non-flowering mock-inoculated plants, plants infected with CMV-Fny and plants infected with the 2b gene deletion mutant of CMV-Fny, CMVΔ2b. The emitted VOCs were distinct from each other when compared by principal component analysis on the relative intensity of ions within the samples . PC1 explained 80.3% of the variation and discriminated between samples from mock-inoculated and CMV-infected plants, whereas PC2 discriminated between samples from mockinoculated and CMVΔ2b-infected plants . Thus, the VOC blend emitted by CMVinfected tomato plants was more distinct from that released by mock-inoculated plants than it was from the volatiles emitted by CMVΔ2b-infected plants. Nevertheless, VOC emission byCMVΔ2b-infected tomato plants was distinct from either mock-inoculated plants or CMV infected plant VOC emission , despite this mutant virus accumulating to markedly lower levels than CMV . Although CMV-infected plants were smaller than either mock-inoculated or CMVΔ2binfected plants, the emission of the combined volatiles on a whole plant basis was similar between mock-inoculated and CMV-infected plants . Indeed, expressing the emission of the combined VOCs by mass of tissue revealed that CMV-infected plants released greater quantities of volatiles compared to mock-inoculated and CMVΔ2b-infected plants . Thus, despite being stunted, CMV-infected plants generated a greater total quantity of VOC than either mock-inoculated or CMVΔ2b-infected tomato plants. Identification by GC-MS of the most abundant VOC by g dry weight of tomato plant tissue showed that terpenoids dominated the profile, with α-pinene, 2-carene, p-cymene, β-phellandrene and the sesquiterpene -caryophyllene being apparent . CMV infection caused quantitative changes in the profile of these VOCs; α-pinene and p-cymene emission increased markedly, whereas 2-carene and β-phellandrene did not, and -caryophyllene almost disappeared from the profile . Isomeric composition was not further determined than that stated here. When VOC emission was compared on a whole plant basis, α- pinene and p-cymene emission rates from CMV-infected plants appeared similar to mockinoculated or CMVΔ2b-infected plants, while 2-carene and β-phellandrene levels appeared to be lower . Bumblebees of a closely related species are known to be repelled by β-phellandrene and 2-carene. Thus, lower emission values of these VOCs from CMV-infected plants may explain why bumblebees displayed an innate preference for CMV-infected tomato plants over mock-inoculated plants in free choice assays . The VOC profiles of mock-inoculated and CMVΔ2b-infected plants were similar, although not identical , and this could explain the bees’ lack of preference in free choice assays . However, optimal self-fertilization requires sonication of the flower to release pollen from the anthers onto the stigma, which is provided either by buzz-pollination from a bee or simulated buzzpollination using mechanical vibration. This is illustrated in Fig 5A, which shows how mechanical buzz-pollination of flowers increased seed production by around a third. Seed production by tomato was very dramatically decreased in plants infected with CMV-Fny to less than 10% of the yield in mock-inoculated plants . Remarkably, artificial buzz-pollination of flowers of CMV-infected plants rescued seed production to a significant degree with seed numbers reaching approximately half the level seen for non-buzzed flowers of mock inoculated plants and about 6- to 7-fold greater than the number of seeds produced in nonbuzzed, CMV-infected plants. The difference in seed yield between mock-inoculated and CMV-infected plants that had been vibrated was less marked than between non-buzzed, mock inoculated and CMV-infected plants . Although CMV-infected plants produced fewer seeds, the mass of individual seeds was unaffected by infection and was not affected whether or not flowers were vibrated . Additionally, the number of flowers produced by CMV-infected plants was similar to the number produced by mock-inoculated plants, and tomato flower morphology was also not markedly altered by infection . Overall plant growth was stunted by CMV infection but, interestingly, virus infection appeared to accelerate the appearance of flowers by a small but statistically significant degree . A recent report indicated that flowers of squash plants infected with the potyvirus zucchini yellow mosaic virus yielded decreased quantities of pollen. However, we found no significant differences in the quantity or viability of pollen released from mock-inoculated and CMV-infected tomato flowers .We investigated the effects of CMV infection on bumblebee-mediated pollination under glasshouse conditions in which the insects could see and interact with flowers .

The bioavailability and tissue distribution of phytochemicals in humans are key factors that need to be clearly established and associated with their biological effects. The fate of phytochemicals in the body, including absorption, metabolism, and distribution, may vary according to the categories of phytochemicals. Ingested polyphenols can be absorbed from the stomach or the small intestine and can undergo conjugation in the intestine and liver to give methyl, glucuronide, and sulfate derivatives . Native polyphenols can also break down, producing smaller phenolic acid derivatives, such as protocatechuic, vanillic, or ferulic acid. These phenolic acids can also undergo phase I and phase II metabolism in the liver . The bioavailability of plant-food bio-active compounds is complex and presents interindividual variation ; however, the extent of such variability and the major determinants involved are currently not established. An example of interindividual variation in the metabolism of plant bio-active compounds is the conversion by the gut microbiota of the soy isoflavone precursors, black plastic nursery pots daidzin and daidzein, to the microbial-derived metabolite equol.

After a soy challenge, 20–30% of Western and 50–60% of Asian populations produce equol. The bacteria involved in the conversion have been identified, but the determinants that govern the daidzein-metabolizing phenotype still have not been fully elucidated. The gut microbiota has also a key role in the metabolism of other plant-food bio-active compounds, such as lignans and ellagitannins . Genetic polymorphisms can also contribute to the interindividual variation in bioavailability. For example, the role of genetic polymorphisms in the interindividual variability in bioavailability of caffeine was demonstrated. Caffeine is mainly metabolized by cytochrome P450 1A2 in the liver, and subjects with the CYP1A2*1F allele variant are considered slow caffeine metabolizers compared with the rapid caffeine metabolizers carrying the wild-type allele . Other factors such as age, sex, and dietary habits may affect the bioavailability of plant-food bio-active compounds. For example, sex differences in the glucuronidation of resveratrol, a polyphenol present in grapes and wine, have recently been observed, which may be explained by sex-specific uridine 59-diphospho–glucuronosyltransferase isoenzyme expression profiles regulated by sex hormones . The existence of an interindividual variability in the bioavailability of plant-food bio-active compounds suggests that there could also exist an interindividual variability in biological response to the consumption of these compounds.

Heterogeneity in the responsiveness to plant bio-active compounds can obscure associations between habitual intakes and health outcomes, resulting in a potential masking of health benefits for specific population groups and thereby limiting our knowledge of the role of the different bio-actives for health. Improving our knowledge of the factors, both genetic and nongenetic [such as age, sex, or genotype], that influence whether plant-food bio-active compounds are more or less effective in individuals will be invaluable to progress in the development of effective and innovative solutions leading to health improvements . However, to date, this interindividual variation in efficacy of plant-food bio-active compounds to modulate physiological outcomes has been little explored. The aim of this review is to provide an overview of the existing studies, both prospective and clinical trials, that has revealed interindividual variability in the responsiveness to the consumption of major plantfood bio-active compounds present in our diet: polyphenols, caffeine, and plant sterols. This review focuses on interindividual variability regarding cardiometabolic outcomes and discloses the potential determinants involved.We identified 6 prospective studies addressing the impact of interindividual variability in biomarkers of cardiometabolic health after habitual intake of a range of different plantbio-active compounds, including coffee and soy .

One area of particular interest relates to the microbially derived soy isoflavone metabolite, equol. In one prospective study, which examined associations between urinary equol excretion, serum lipids, and carotid intima thickness in 572 Chinese participants, 25% were equol excreters on their usual diet. In relation to other characteristics, the number of equol producers was similar between men and women, and there was no significant difference between equol-producer phenotype and age, dietary intakes, blood pressure, or BMI . Equol excreters had significantly lower TG and IMT levels compared to non-equol excreters . Although there was no association between soy isoflavone intake and serum lipids or IMT in the non– equol excreters, equol excreters within the highest quartile of intake had significantly lower IMT and higher HDL cholesterol concentrations than those in the lowest quartile of soy intake. Although this was an Asian population, habitual intakes of isoflavones were low, with a mean intake of 13 mg/d in both the equol- and non–equol-producer groups . The findings are therefore intriguing because data from the extensive literature on soyintervention studies suggest that an isoflavone intake >25 mg/d is required for any biological or clinical effect . The lack of an effect of isoflavone intake on CVD risk in women from the EPIC population was therefore not surprising, given that the median intake of isoflavones was only 0.4 mg/d. This study did not assess equol-producer status, and there was no difference in the association between habitual isoflavone intake and CVD risk when stratified by smoking , BMI, hormone replacement therapy use, age at intake, and hypercholesterolemia . This prospective study also examined associations between habitual lignan intakes and CVD risk in women and observed no association with intake , although the authors suggested a decreased risk of developing CVD in participants who were past smokers and had a higher habitual lignan intake. Therefore, available data on soy and the microbially derived metabolite equol are very limited. The impact of the equol-producer phenotype requires further investigation in population groups in which there is a wide variability in intakes in order to more carefully examine the magnitude of interindividual variability in response to biomarkers of cardiometabolic health and particularly the importance of the microbially derived metabolite equol. Four prospective studies have examined the impact of several factors in explaining the association between coffee intake and CVD risk . Whether polymorphism in the CYP1A2 gene, coding for the main enzyme responsible for the metabolism of caffeine, modulates the association between coffee intake and risk of CVD and related biomarkers was addressed in 3 studies. In one study, the risk of hypertension associated with coffee intake was shown to vary according to CYP1A2 genotype, with carriers of the slow-metabolism *1F allele at increased risk with higher coffee intake but not participants with the fast metabolism *1A/*1A genotype .

In a more recent study from this same hypertensive cohort, the association between coffee intake and impaired fasting glucose was stronger in carriers of the *1F variant, with the highest risk in heavy drinkers [$4 cups/d ] . In relation to myocardial infarction, 30 plant pot in a case-control study coffee intake was only associated with an increased risk of nonfatal myocardial infarction among participants with slow-caffeine metabolism . Only one study examined whether the relation between coffee intake and incident of coronary artery disease is dependent on the metabolism of catecholamines, specifically polymorphisms of the catechol-Omethyltransferase gene. In a cohort of 773 men, the relation between consumption of caffeinated coffee and the incidence of fatal and nonfatal CAD was dependent on COMT genotype. In men who were either homozygous for the high-activity COMT allele or heterozygous, substantial coffee intake did not increase the incidence of acute coronary events. However, for those who were homozygous for the low-activity COMT allele, heavy coffee consumption was associated with a higher incidence of acute coronary events, and the relative CAD incidence was >200% higher among drinkers of >6.5 cups of coffee/d after multivariable adjustment . Taken together, these few prospective studies have shown that there is interindividual variability in response to the consumption of plant-food bio-active compounds and that individuals do not equally benefit from the consumption of these phytochemicals. Different determinants, such as gut microbiota, genetic polymorphism, or smoking, have been suggested to be involved in these between-subject variations. It should also be noted that coffee is a source of not only caffeine, the amount of which can vary depending on brewing , but also of other micronutrients, such as chlorogenic acid, which has been shown to mediate the blood pressure rise caused by coffee intake .Age is the strongest independent cardiovascular risk factor for CVD, as indicated in most methods of risk scoring, such as the Framingham risk score or the European Society of Cardiology SCORE system . Aging is also associated with increased vascular stiffness, endothelial dysfunction, and isolated systolic hypertension . All these age-associated changes in the vascular system are known to have an effect on the bioactivity of some drugs, such as verapamil, albuterol, or benzodiazepines , and potentially could also have an effect on the bioactivity of plant-food bio-actives, which undergo the same conjugation pathways when absorbed.To date, few studies have examined the effects of age on the cardiometabolic effects of food bio-active compounds . Three studies have investigated age-dependent effects of cocoa flavanols on vascular function , with conflicting results. However, only one of them was a controlled study specifically designed to investigate the effects of flavanols in the context of the aged cardiovascular system. A double-blind RCT demonstrated that consumption of a flavanol-rich drink 2 times/d for 2 wk reversed age-related increases in blood pressure together with vascular stiffness in healthy elderly men. CF-intake–associated improvements in the compliance of large arteries were complemented by a decrease in pulse wave velocity and aortic augmentation of systolic blood pressure . Endothelial function in large conduit arteries was also significantly improved in healthy young and elderly individuals. These beneficial effects were associated with an improved dilatory capacity of resistance arteries, lower diastolic blood pressure , and increases in microcirculatory perfusion and RBC deformability. Cardiac output was not affected by CFs. Importantly, despite age-dependent differences in baseline flow-mediated dilatation , PWV, and DBP, the magnitude of the changes in the vascular response to CFs was not significantly different between the young and the elderly. In contrast, flavanol consumption improved only SBP and the augmentation index in the elderly group . This is probably because SBP is slightly higher in the elderly, mainly caused by stiffer arteries. Plasma concentrations of flavanol metabolites were not significantly different between young and elderly individuals, suggesting that differences in bioavailability could not explain the differences observed in biological responses. Of note, endothelial dysfunction is a well-established response to cardiovascular risk factors and precedes the development of atherosclerosis. The measurement of ultrasound-based endothelium dependent FMD in the brachial artery is the more widely used noninvasive measure of endothelial function and constitutes a clinical surrogate marker of vascular health . This technique consists of assessing the change in the diameter of the brachial artery after the increase in shear stress induced by a reactive hyperemia, with the degree of dilatation reflecting arterial endothelial NO release . The aortic AIX is closely related to wave reflections and constitutes a surrogate marker of arterial stiffness . In agreement with previous data, a recent study showed that the absorption, distribution, metabolism, and excretion of CFs was not significantly different between young and elderly healthy subjects after consumption of a similar amount of CFs . However, small but significant differences in metabolism were reported at a higher intake amount of CFs , with higher glucuronidation, lower methylsulfation, and lower urinary excretion of gut microbial g-valerolactone metabolites observed in the elderly. This observation suggests that dose-response studies covering the amounts of bio-active intake that can be achievable through a normal diet are necessary when investigating the interindividual variability in the ADME of plant-food bio-active compounds. A study also investigated whether the consumption of a flavanol-rich cocoa drink could improve blood pressure and endothelial function in healthy young and elderly men . No changes in blood pressure or endothelial function were observed in any group after 4–6 d of daily CF consumption. However, an effect on the last day of the study was seen in both groups after 90–180 min of CF consumption, and when compared with baseline values of day 1, the effect was higher for the elderly volunteers. Pulse wave analysis showed a similar pattern, with higher vascular responses in the elderly after acute consumption. The authors attributed these effects to an increase in NO production because responses to the endothelial NO synthase inhibitor L-nitroarginine-methyl-ester were also greater in the elderly. Nevertheless, the relevance of comparing changes in vascular function after acute consumption on days 4–6 with baseline levels on day 1 remains to be established.