Gollin et al. explain that the Green Revolution “emerged from philanthropic efforts … to address the challenges of rural poverty and agrarian unrest in the late 1950’s and early 1960’s, and it involved a concerted effort to apply scientific understandings of genetics to the development of improved crop varieties that were suited to the growing conditions of the developing world.” Based on longitudinal international research and impact models from 1960-2000 , Evenson and Gollin suggested that the Green Revolution considerably raised the health status of preschool children and lowered the infant and child mortality rates in developing nations. In his retrospective review of the Green Revolution, Pingali demonstrates that Green Revolution “contributed to widespread poverty reduction, averted hunger for millions of people, and avoided the conversion of thousands of hectares of land into agricultural cultivation.” However, these successes were accompanied by unintended negative consequences . In the post-Green Revolution, Pingali shows, food insecurity persists, nutrition is lagging, and environmental impacts were mixed. Pingali characterizes these costs as unintended negative consequences, not because the technology was bad, but because of the policies that encouraged rapid implementation these technologies. Policies led to interregional food security disparities in South Asia and South America , a lackof support for women in agricultural technology transfer , the abandonment of micro-nutrient rich traditional crops for incentivized staple crops , and the over-use of inputs such as fertilizers, pesticides, and irrigation water . Food insecurity, square flower bucket as defined by the New Oxford American Dictionary , is the state of being without reliable access to sufficient quantity of affordable, nutritious food.

Food insecurity is an ongoing matter with an increase in world undernutrition since 2015 to 821 million people, and a long-continued global increase in micronutrient deficiency among the non-hungry that leads to obesity . Regarding addressing food insecurity in the long-term, the United Nations Food and Agriculture Organization argues that “global resources are sufficient,” estimating enough food can be produced for the forecasted peak population of 9.1 billion people. Instead, the FAO argues that food security is limited by economic and institutional frameworks in the distribution of the available food stores. The FAO argues that food security is also limited by local resource constraints and the overuse of resources until a tipping point is reached and the society becomes impoverished. Obesity and other effects from improper nourishment are most prevalent in North America . In 2006, the USDA reported that 7.4 million acres of additional cropland would need to be harvested for Americans to eat the nationally recommended diet because the domestic food system at the time did not produce enough fruit, vegetables, or dairy . Furthermore, the they indicated that the agricultural industry over-produced grains, but American’s were still receiving too few whole grains because much of it was being consumed as refined-grains. The report suggested that grain production could decrease by 5.4 million acres and, with replacing refined grains with whole grains, still have enough grain to satisfy the recommended US Diet. More recently, the USDA shows that MV of corn, wheat, rice, and soy are still over consumed and fresh fruits and vegetables are under-consumed and have been implicated in the obesity epidemic . Scientists and journalists have long implicated governmental subsidies and other food-aid programs in the cause of obesity and improper nourishment , but others have refuted this point .

Obesity linked to an unbalanced diet is also growing globally. In many developing nations, displacing small, multi-crop, and non-staple farms with large monoculture staple farms diversifies food availability and changes local diet . For many people, the change in diet has been reduction in diet diversity, which perpetuates micronutrient deficiency . People who experience such a change in their food system undergo a “nutrition transition” to refined foods high in fat, sugar, and salt which lead to obesity , disproportionality affecting financially insecure people because traditional varietals now have a higher market price . Other nations that are experiencing large socio-economic changes suffer from the “double burden of malnutrition” , in which financially insecure people are undernourished and financially secure people areobese. In this case, those who are obese now have money and access to food, but cannot productively process that food because they are metabolically adapted to hunger from their youth when they were undernourishment . Policies across the world encouraged farmers to transition into growing MV crops for their high productivity . Globally, MV seeds and infrastructure, such as irrigation and tractors, are respectively expensive to buy and operate while the products have a comparatively low market value . The upfront cost is too great for small farms during the transition from subsistence farming; they often go into debt and then lose farms because they are not able to turn a large enough profit . In the US, instead of institutions that foster personalized connections between farmers and consumers that support medium and small diversified farms, what crops are grown and how they are grown is largely dictated by governmental subsidies, loan structures, and insurance .

These policies shape operating costs in such a way that helps only large farms succeed in turning a profit . In the United States, small and medium sized farms are shrinking in number, and large farms are growing in space, production, and profits . The number of farms has reduced from 7 million in 1935 to 2 million in 2016 , with 2.2% of farms controlling over a third of all crop land . In 2015, most farms operated above the US-median house hold income of $56,516, but many supplemented their household income with income from off-farm sources . For the farms accruing less than $10,000 in sales, which accounts for 48% of all farms, farm production had a median negative effect on household income, meaning the farms operated at a financial loss . In contrast, farms that made over $1,000,000 in sales had a median household income of over $300,000 from farming alone. Developing nations transitioning to industrial agriculture are also experiencing trends of increasing economic disparity among farmers 2010. Researchers and activists often characterize modern industrial agriculture as unsustainable because of its contributions to global climate change and other forms of environmental degradation. Clearing forests for crop land leads to changes in the hydrological cycle and reduction in carbon sequestration . Modern industrial agriculture creates up to 25% of global anthropogenic greenhouse gases from preproduction processes , production direct and indirect emissions from agriculture, and post production processes . Scholars also implicate modern industrial agriculture in reducing ecosystem health and diversity. Applied pesticides and fertilizers contaminate the water, soil, and air, poisoning humans, animals, and microorganisms .Excess nitrogen in soil reduces plant diversity and reproductive success . Certain pesticides have been proven to cause cancers in animals and linked to cancer cases and other health concerns in humans . Particular pesticides have been linked to a long term decline in bird and beneficial insect populations , including honey bees . Agricultural run-off in the Mississippi River created ecological “dead zones” in the Gulf of Mexico , contaminates large bodies of fresh water with blue-green algae blooms like the 2018 Lake Okeechobee algae bloom , and may exasperate naturally occurring red tides like the 2018 Florida red tide . Modern industrial agriculture also exhausts the natural resources it needs to function. It depletes accessible groundwater resources, which takes thousands of year to recharge, for irrigation and degrades soil, which is also a precious and difficult to rebuild resource, through poor farming practices such as seasonal tillage . These global changes strain the efficacy of all forms of food and agricultural systems, from small farm families to large industrial farming organizations. The agriculture industry suffers from ongoing significant declines in crop and livestock production from climate change induced stresses; societies are struggling with availability of food and water during intensifying droughts; humans are facing food, water and vector borne disease .Neither activists nor researchers share a canon definition or vision of a “sustainable” food and agriculture system, because these are formed by varying views and political climates . Agroecologist David Cleveland argues that the term “has been used to mean everything from giant, black flower bucket laser-level fields of genetically engineered soybean to tiny hillside plots growing tumbles of traditional maize, bean, squash, and herbs, cultivated by hand” . Mary V. Gold of the USDA National Agricultural Library suggests that “sustainable agriculture” is a term that defies definition but provides “a sense of direction, and an urgency, that has sparked innovative thinking the agricultural world.” However, there are common themes among the many conceptualizations of sustainable agriculture .

The first, perhaps most common, theme is to feed humans a nutritional diet for a long or indefinite amount of time . The second theme is that “sustainable” agricultural system must maximize environmental, social, and economic factors to achieve the goal of feeding people for a long, long time 1990. The third theme is that agriculture needs to be reframed as an intertwined natural and human system, typically called an agroecosystem .Gliessman defines an agroecosystem as a site or integrated region of agricultural production understood as an ecosystem . When considering levels of ecological organization , Gliessman’s interpretation of an agroecosystem is equivalent to an ecosystem composed of communities of living organisms and their environment, including all abiotic factors. In practice, an agroecosystem exists in and is inseparable from the spatially and ecologically larger landscape. Natural ecosystems and agroecosystems exist on a continuum of degree of human influence, where few, if any, natural ecosystems are completely void of human influence and agroecosystems vary in the degree of their human influence. Conceptually, boundaries of an agroecosystem are somewhat arbitrary because, as Gliessman states, “an agroecosystem is enmeshed in both social and natural worlds” . However, in terms of management, there is typically a defined spatial boundary, like a farm. Anything that comes from off the farm is an external human input, apart from natural inputs like sun light. All things on the farm are a part of the agroecosystem and are managed, whereas things beyond the boundary are a part of the natural ecosystem.Conway and Barbier describe agroecosystems as “a hierarchy ascending from the level of the individual plant or animal all the way to national systems linked by international trade” . Their hierarchy differs from Gliessman’s in that it is not a literal comparison to that of a local ecosystem. Instead, it expands Gliessman’s concept of an agroecosystem by incorporating the social constructs of the agrifood industry. This conceptualization of agroecosystems necessitates that each level “be analyzed and developed both in its own right and in relation to other levels above and below” . In this view, sustainable agricultural development cannot happen from farm-level research nor macro-economic policy alone. Ecosystem Services. Like natural ecosystems, agroecosystems systems provide ecosystem services . The researchers that complied the Millennium Ecosystem Assessment define ecosystem services as “the benefits that people obtain from the ecosystem” . These benefits include provisioning services, regulating services, cultural services, and supporting services . For a comparison of Millennium Ecosystem Assessment’s categorization of ecosystem services to other categorizations, see . By utilizing and generating ecosystem services, sustainable agriculture can reduce or eliminate the use of resources that contribute to a significant percentage of greenhouse gas emissions, , resources that poison consumers , and resources that decline through crop production .Another characterization of sustainable agriculture, particularly among activist groups like many permaculture communities, is “local.” Local agriculture can increase community resource security, public health, nutrition, social capital, and microenterprise opportunities . Not only food, but many essential goods used today like timber,fiber, medicine, household cleaners, etc., are or can be derived from local agricultural resources or byproducts. Growing resources locally helps a community achieve a certain amount of control over what they will consume, and the agricultural system that produces it – i.e., food and resource sovereignty . Locally grown resources also reduce or eliminate energy and other materials used in domestic and international transportation and storage, however, this is typically a very small percentage of a farm’s total fossil fuel use and greenhouse gas emissions . Transitioning to local agriculture and food systems in part addresses the underlying issue that people do not have much power over the food they eat or standards for how it is produced.

Every interaction is an opportunity to build your market. Lecture 2 also addresses the issue of on-farm events. If you do a good job of engaging your community through social media and when you are out and about, then it’s likely your community will be interested in seeing your farm and learning about what you do. You might find that visitors drop by for an impromptu farm tour. This can be difficult to manage, especially if there are very few workers on your farm. Hosting an on-farm event is a popular way of sharing all that is beautiful and interesting about your farm while creating a sense of community amongst all the different people who may be interested in coming out to learn more about how their food is grown. On-farm events can be a lot of fun, and regardless of whether they make money, they may generate good will in the community. But as discussed in Lecture 2, it’s important to be aware of the potential downside: events take a great deal of time and effort, aren’t always profitable, and you can open yourself up to new liabilities when you open your farm to the public.Phenylpropanoids are specialized metabolites involved in several aspects of plant growth and development and in the responses of plants to environmental stimuli. These compounds are synthesized from key intermediates of the shikimate pathway, which are structurally modified by the combined activities of lyases, transferases, ligases, reductases and oxygenases, flower bucket resulting in the organ- and developmental-specific synthesis and accumulation of diverse metabolites . The phenylpropanoid pathway provides the building blocks for lignin, suberin, and condensed tannins that play a role in structural support and mechanical strength.

Lignin is a major contributor to feedstock recalcitrance and negatively affects the conversion of plant biomass into downstream products in bio-refineries . Further, this pathway is key for the production of anthocyanins for organ pigmentation, flavonols and flavones for UV protection, various flavonoids and isoflavonoids for plant-microbe interactions, and antimicrobial phytoalexins for protection against pathogens . In addition to their biological functions in planta, phenylpropanoids are economically important metabolites. They constitute important components in the human diet, acting as nutraceutical compounds with antioxidant, chemopreventive, antimitotic, neuroprotective, cardioprotective, and anti-inflammatory activities. Several phenylpropanoids are considered high-value biochemicals employed in the production of fragrances, pharmaceuticals and biopolymers . Systems biology approaches have enabled the characterization of numerous aspects of the phenylpropanoid metabolism in different plant species, including genes, enzymes and metabolites involved in the different branches of the pathway and how these branches are regulated. However, there is still much to be explored and determined in terms of regulation, de novo biosynthesis, transport, compartmentation, and polymerization of phenylpropanoids. In addition to their complex biosynthetic machinery and extensive chemical diversity, the synthesis and accumulation of metabolites is also largely dependent on the tissue type, developmental stage, plant species, or might be triggered in response to specific environmental conditions . Advances in “omics” technologies provide a timely opportunity to further characterize even subtle changes in the levels of transcripts, enzymes and metabolites, and thus to provide a comprehensive systems view of phenylpropanoid metabolism throughout plant development and during stress responses.

Furthermore, phenylpropanoid bioengineering holds promise to generate more resilient and nutritious crops, to maximize our arsenal of useful biomolecules, and to re-design high-yield and sustainable bioenergy feedstocks by means of biotechnology. This Research Topic aimed to gather recent findings in all aspects of phenylpropanoid metabolism gained by means of systems biology approaches and the utilization of biotechnologyto exploit the economic, medicinal and nutraceutical potential of phenylpropanoids. The topic is organized into four sections: structural, molecular and computational approaches toward unraveling the biosynthetic pathways involved in synthesis of diverse phenylpropanoid-derived metabolites; discovery of genes and/or gene networks involved in distinct aspects of phenylpropanoid metabolism via omics technology; functional characterization of genes involved in the phenylpropanoid metabolism and its coordination with physiological processes; and biotechnological approaches to exploit the economic, medicinal, and nutraceutical potential of phenylpropanoids.The chemical diversity of phenylpropanoids results from the modification and amplification of a set of core structures derived from the shikimate pathway. A vast array of regulatory proteins, biosynthetic enzymes, oxidases and other genes are recruited to produce the various classes of phenolic metabolites. Additionally, many phenylpropanoids are specific to just one or a few plant species, underscoring the complexity of phenylpropanoid biosynthesis and the need for comprehensive characterization studies in diverse species that expand our knowledge base beyond traditional model plant and crop species. Structural, molecular and computational approaches have been applied to identify genes, enzymes and metabolites involved in the biosynthesis of phenylpropanoids in different plants. Delli-Ponti et al. have reviewed how gene expression and co-expression networks can be used as tools to uncover specialized metabolism biosynthetic pathways.

Also using a computational approach, Elder et al. have applied density functional theory calculations to evaluate the thermodynamics of coupling modes and subsequent rearomatization reactions between coniferyl alcohol and hydroxystilbene glucosides, which has been detected as a natural monomer in the bark lignin of Norway spruce. To this end, the effect of chilling treatment on the accumulation of phenylpropanoids and on antioxidant activity in seedlings of two rice varieties was studied by Du et al. . Lignin is a phenolic polymer important for plant growth and development but it is also considered a major bottleneck to the efficient conversion of plant biomass into downstream products. Rosado et al. have reported an indepth characterization of the structural characteristics of lignins present in rice husks and straw, which are agricultural byproducts that can be used to produce chemicals and materials in biorefineries. To identify the timing and key parameters of cell wall recalcitrance across different switchgrass genotypes, Saha et al. measured cell wall composition and phenylpropanoid/lignin biosynthesis gene expression in three switchgrass genotypes representing lowland and upland ecotypes. Yao et al. reviewed recent progress in defining the lignin biosynthetic pathway in lycophytes, monilophytes, gymnosperms, and angiosperms, and integrated new insights on major transcriptional regulators. In another study with evolutionary implications, Rencoret et al. structurally characterized the lignin-like fractions isolated from several ancestral plants, including those from moss, lycophyte, horsetail, fern, cycad, and gnetophyte species. Blaschek and Pesquet provided an overview of the differences and similarities in the structures, reaction mechanisms, substrate specificities, and functional roles between phenoloxidases. Because grasses are able to synthesize phenylpropanoids from either phenylalanine or tyrosine , Simpson et al. employed 13C isotopic-labeled precursors and mass spectrometry-based metabolomics to determine the downstream metabolites derived exclusively from Phe and Tyr in sorghum. Several phenylpropanoids show bioactivity that might influence plant growth and development or might be beneficial for human health. El Houari et al. reviewed reports describing altered accumulation of bioactive phenylpropanoids as the causal factor for observed phenotypes of lignin mutants in Arabidopsis. Cappellini et al. reviewed the recent progress in understanding the anthocyanin biosynthetic pathway in plants, with special emphasis on the differences in molecular mechanisms between monocot and dicot plants, and discuss the biological activities of anthocyanins as beneficial components of the human diet. Similar to anthocyanins, tannins form another group of phenolic compounds with beneficial effects on human health. Wang et al. performed a genome-wide analysis of the tannase gene family to identify candidate genes responsible for tannin metabolism in three nut tree species in the Juglandaceae family: walnut, pecan, and Chinese hickory.The identification of genes and transcriptional networks responsible for specific accumulation patterns of phenylpropanoids during a physiological development process or a stress response is essential to elucidate and harness the fine regulatory mechanisms involved in these patterns. Recent advancements in omics technologies enable integrated approaches to unravel these mechanisms at the transcriptomic, proteomic, plastic flower bucket and metabolomic levels. These studies provide platforms to guide future research on improving crops for human health and wellness. Tang et al. leveraged single-molecule real-time sequencing technology to elucidate flavonoid synthetic pathways in blueberries. Their transcriptome analyses led to the discovery of a R2R3 MYB transcription factor that can positively regulate anthocyanin synthesis in fruits. 5-aminolevulinic acid is a plant growth regulator that induces fruit coloration and thereby finds potential applications in modern fruit production. A transcriptome study by Zheng et al. identified the differentially expressed genes associated with ALA-induced anthocyanin accumulation in apple, including two R2R3-MYB transcription factors involved in flavonoid accumulation. A study by Aniciˇ c et al. ´ investigated flavonoid metabolism during fruit development in rockrose, a traditional medicinal plant rich in bioactive phenylpropanoids, using comparative metabolomic and transcriptomic approaches. This work highlights correlations between expression patterns of biosynthetic genes and the content of proanthocyanidins.

Phenolic compounds are modulated by biotic and abiotic stresses, and a study by Laoué et al. used quantitative trait locus mapping and RNA-Seq to explore the complex polygenic network underlying the constitutive production of specific stilbenoids, flavonoids, and lignans in white spruce. Understanding the formation of secondary cell walls and their lignification has important agro-industrial applications. Hixson et al. , by undertaking an integrated analysis of the metabolome, transcriptome, and proteome of Arabidopsis lines mutated in arogenate dehydratase genes, exposed the involvement of novel proteins and additional post-transcriptional and translational processes that govern phenylpropanoid/lignin biosynthesis. As a proxy to study SCW formation in the bioenergy crop sugarcane, Simões et al. established a lignifying cell culture system that they probed with transcriptomic and metabolomic analyses to illuminate the molecular mechanisms involved in this differentiation process, leading to the discovery of regulatory modules that control SCW deposition.The phenylpropanoid pathway in plants plays a major role in the synthesis of a wide variety of secondary metabolites. Metabolites originating from this pathway are frequently involved in plant structure or chemical signaling and defense, including flavonoids, lignins, hydroxycinnamic esters, flavonoids, anthocyanins and tannins. Dietary flavonoids, anthocyanins, proanthocyanidins, hydroxycinnamoyl acid amides and lignans are bioactive compounds that have been shown to exhibit multiple health promoting and antioxidant activities. Lignans are plant secondary metabolites composed of a core scaffold that is formed by two or more phenylpropanoid units that can adopt a spectrum of different structural forms. Chen et al. identified two non-selective uridine diphosphate glycosyltransferases from Isatis indigotica Fort. that catalyze the addition of a sugar molecule onto several structurally diverse lignin acceptor substrates. Shi et al. sought to explore the transcriptional regulatory mechanisms of anthocyanin and proanthocyanidin biosynthesis in Chinese bayberry, of which the fruit is considered an important dietary source of natural antioxidants. They identified a MrMYB6 gene that is highly upregulated during the latter stages of fruit development and determined it is a negative regulator of anthocyanin and proanthocyanidins through formation of a complex with two transcription factors, bHLH and WD40. Busche et al. carried out a study five 2-oxoglutaratedependent dioxygenases involved in the formation of the flavonoid aglycon in banana : flavanone 3-hydroxylase, flavonol synthase and anthocyanidin synthase. Biochemical analysis of several recombinant candidate proteins showed that MusaF3H1 and MusaF3H2 act as flavanone 3-hydroxylases, MusaFLS1 and MusaFLS3 both function as flavonol synthases, and MusaANS has anthocyanidin synthase activity. Elucidating the activity of these genes will facilitate the development of bananas with higher nutritional value. Hydroxycinnamoyl acid amides, such as clovamide, are phenylpropanoid metabolites that play roles in protecting plants from biotic and abiotic stresses. Sullivan and Knollenberg identified, cloned and biochemically characterized a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase from red clover that is capable of synthesizing clovamide and related hydroxycinnamoyl amides in vitro. Characterization of this enzyme activity expands our knowledge of the poorly characterized family of BAHD hydroxycinnamoyl-CoA transferase enzymes and will aid in future studies aimed at understanding the molecular basis of substrate specificity within this important family. Lignin is a heterogeneous phenolic polymer that is highly abundant in the secondary cell walls of all land plants and is composed of three major monolignol subunits: 4- hydroxyphenyl , guaiacyl , and syringyl . The monolignol building blocks of lignin are synthesized by enzymes acting in concert that catalyze sequential reactions. Lin et al. provide direct evidence that two key enzymes involved in monolignol biosynthesis, 4-Coumaric acid:CoA ligase and 4-hydroxycinnamoylCoA:shikimic acid hydroxycinnamoyl transferase, form a Ptr4CL-PtrHCT complex in Populus trichocarpa and its formation is a potential mechanism to modulate metabolic flux during secondary cell wall synthesis. The brown midrib phenotype found across several C4 grasses has been critical for identifying mutants compromised in lignin synthesis. Tetreault et al. used a combined bulk segregant analysis and nextgeneration sequencing approach to show that bmr30 encodes a chalcone isomerase and is involved in synthesis of the flavonoid tricin and not a monolignol.

Annual populations exhibit clinal variation in phenological, physiological, and vegetative traits that reflect climatic variation in the length of the growing season , whereas perennial populations from coastal, inland, and montane habitats comprise separate ecotypes with consistent phenotypic differences . Coastal and inland perennials overwinter as vegetative rosettes produced from above-ground stolons , whereas montane perennials primarily overwinter as below-ground rhizomes . Given extensive phenotypic variation in these and other traits and incomplete reproductive barriers, taxonomists have variously recognized between 4-20 species in the M. guttatus complex . Recent genomic evidence suggests extensive introgression among ecotypes, reflecting the recent origin and /or on-going gene flow among taxa in this complex .Direct selection – The strength and pattern of direct selection varied among ecotypes, years, and fitness components . In general, fitness components related to fecundity showed consistently positive relationships with flowering stem diameter and flower size across ecotypes and years . Conversely, selection on flowering time via fecundity contributions varied from positive to negative among years and ecotypes,though selection through ovule number was always positive whereas selection through fruit number was always negative . Within perennial ecotypes, direct selection through rosette production was only detected in 2012 . In contrast to the pattern for fecundity selection, selection on flowering stem diameter through rosette production was negative in montane perennials, though this was only marginally significant . In inland perennials, wholesale plant containers later flowering and larger seedlings increased rosette production, though selection on seedling size was only marginally significant .

In 2012, inland perennials only experienced selection through rosette production, whereas in 2013 all selection in this ecotype occurred through fecundity . There was no significant relationship between adult overwinter survival and any of the four traits examined in either year. Integrated selection – Integrating selection by estimating trait elasticities altered the strength of selection relative to estimates through individual fitness components. For most traits, integrated selection was weaker than direct selection because individual fitness components are weighted by their elasticities, which are necessarily less than one. This effect was most pronounced in perennial ecotypes because fitness component elasticities were decreased in perennials relative to annuals, which exhibit high elasticities for fecundity . Large selection gradients in perennial ecotypes occurred through fitness components with smaller elasticities . Correspondingly, integrated selection on flowering time was generally weaker in perennials relative to annuals , even though direct selection gradients through individual fitness components were larger in perennials . In one case, integrated selection was stronger than direct selection because selection occurred in the same direction through multiple fitness components. Robust annuals experienced selection for larger flowering stem diameter through both fruit number and ovule number per fruit in 2013 , resulting in strong integrated selection for larger stems . A similar pattern occurred in 2012 and for larger flowers in inland perennials in 2013 , though in both cases selection through fruit number was only marginally significant. Total integrated selection – Correlations among traits had diverse effects on the strength of selection . Positive correlations among traits increased the strength of selection in some cases. In fast-cycling annuals, for example, selection for increased flowering stem diameter caused correlated selection for larger flowers and later flowering in both years .

Similarly, positive correlations between flowering stem diameter and flowering time increased the strength of selection on both traits in robust annuals in 2012 . In other cases, correlations among traits opposed the direction of selection on individual traits, weakening the strength of selection overall. For example, selection for earlier flowering in robust annuals in 2013 was opposed by a positive phenotypic correlation between flowering time and flowering stem diameter combined with selection for larger flowering stems . Similarly, selection for larger seedlings in fastcycling annuals in 2013 was opposed by a negative correlation between seedling size and flowering time combined with selection for later flowering . In one case, multiple trait correlations opposed each other, effectively canceling out any effect of correlated selection on stem diameter in robust annuals in 2013. In this example, a positive correlation between stem diameter and flower size combined with selection for larger flowers was opposed by a positive correlation between stem diameter and flowering time combined with selection for earlier flowering . These opposite and nearly equal effects resulted in an estimate of total integrated selection very similar to the strength of integrated selection on stem diameter alone .Although divergent life history strategies within M. guttatus have become a model system for ecological genomics and speciation, the selective forces maintaining life history variation have not been well characterized in perennial populations. The adaptive significance of rapidly developing genotypes in seasonally drying habitats is intuitive, yet evidence that a perennial life history strategy is adaptive in habitats with greater water availability remains scarce. Although coastal perennials are locally adapted to coastal habitats relative to annuals, this appears to be largely mediated by increased tolerance to salt spray, and thus greater seedling survival and fruit set, rather than an advantage of perenniality per se . In this study, I directly compared the demographic performance of annual and perennial ecotypes within a montane perennial habitat and tested how specific transitions in the life-cycle contribute to differences in performance using LTREs.

With this approach, I found mixed evidence for local adaptation. A significant effect of ecotype on overall performance, combined with non-significant effects of population within ecotype, support that there are real differences in life history strategy and performance among these a priori ecotypes. I found some evidence that a perennial life history strategy is adaptive in a perennial habitat, since inland perennials consistently had the greatest demographic performance whereas fast-cycling annuals had the lowest. Further, transitions associated with adult survival and vegetative growth contributed positively to λ in the montane perennial ecotype, whereas fecundity transitions contributed negatively. However, there was no evidence that the montane perennial ecotype is locally adapted since inland perennials and robust annuals outperformed montane perennials in both years. One potential explanation for the relatively poor performance of themontane perennial ecotype is that the importance of below-ground rhizomes was not fully captured during the time-frame of this study. Indeed, montane perennial genotypes invest extensively in below-ground rhizomes and may be able to persist during harsh conditions when other strategies lacking below-ground structures would become extinct. Natural recruitment plots suggest that seedling recruitment is low relative to rosette recruitment. Longer-term monitoring of the relative performance of seedlings and rosettes under a range of environmental conditions, including drought, competition, and frost, would refine estimates of demographic performance. Alternatively, the low performance of the montane perennial ecotype could be due to genetic load, given extensive clonal growth and low flower production and seedling recruitment. Within the montane perennial ecotype, I did find some evidence for local adaptation as the native Eagle Meadows population outperformed both foreign populations, although this difference was not significant.As expected, life history ecotypes exhibit different fitness strategies even within a common montane environment. The performance of annual ecotypes is influenced most strongly by seed production, whereas perennial performance is also achieved through the growth and survival of clonal rosettes. Among perennials, montane populations produce few flowers and rely extensively on clonal growth through below-ground rhizomes. This divergence in the relative importance of specific fitness components influences the net pattern of selection. Interestingly, the direction of fecundity selection was similar across life history ecotypes, consistently favoring greater allocation to reproductive structures including larger flowers and larger reproductive stems. Conversely, selection through clonal growth, when detected, favored smaller reproductive stems and seedlings . These contrasting effects of individual fitness components resulted in contrasting selective landscapes for different life history ecotypes even within a common environment. In particular, integrated selection tended to be weaker in perennials relative to annuals due to the reduced elasticity of any one fitness component. A major goal of evolutionary biology has been to document the strength of selection in wild populations. In a review of mean-standardized selection estimates, Hereford found surprisingly strong directional selection through individual fitness components and suggested that these are unlikely to represent the strength of selection in general. One potential explanation is trade-offs among fitness components in the direction of selection, plastic pot manufacturers yet evidence for such trade-offs is mixed and based on relatively few studies . I only detected concurrent selection on the same trait through multiple fitness components for stem size and marginally for flower size. In these cases, selection through multiple fitness components increased the strength of integrated selection, because underlying selection gradients through fruit and ovule number were in the same direction. These traits are directly related to overall investment in reproductive biomass; such “size” traits may frequently be positively correlated and increase multiple components of fitness due to environmental covariance . I found some evidence for conflicting selection among fitness components in comparisons across ecotypes and years.

Later flowering consistently increased ovule production but decreased fruit production, whereas larger stems increased fecundity but decreased rosette production. However, I never detected these effects within the same ecotype and year, so it is unclear whether these trade-offs act simultaneously to decrease the strength of selection. Even if selection on a given trait primarily occurs through a single fitness component, life history divergence will still affect the strength of selection. Failing to integrate multiple fitness components will bias the strength of selection, since individual fitness components contribute unequally to population growth among environments and life history strategies . Integrating selection incorporates this variation by multiplying selection gradients by the elasticities of their respective fitness components. Since elasticities are necessarily less than one, integrated measures of selection will always be weaker than selection gradients, unless multiple fitness components are positively correlated. Thus, previous conclusions about the average strength of selection in wild populations are likely overestimates . The strength of selection gradients depends in part on the degree of fitness variation, however demographic reviews have found that the fitness components with the largest elasticities are often the least variable within populations . In this study, I found that strong selection gradients in perennials occurred through fitness components with smaller elasticities, weakening the strength of integrated selection relative to annuals. Large selection gradients may reflect less important fitness components, rather than strong phenotypic selection, and this potential bias should be greatest in long-lived iteroparous organisms where some fitness components will only contribute weakly to total fitness.Incorporating phenotypic correlations among traits frequently altered the strength of selection. Although phenotypic correlations determine the strength and pattern of selection, inference about the evolutionary response to selection requires an understanding of the underlying genetic variance-covariance structure. However, there are several lines of evidence to suggest that the phenotypic correlations observed here reflect genetic correlations within or among ecotypes. First, the randomized plot design used in this experiment minimizes the potential for environmental effects to generate trait correlations while the use of highly divergent ecotypes and multiple populations within each ecotype maximizes the potential for genetically based trait variation. Second, previous research has shed light on the genetic basis of many traits in this species. Below, I explore how selection through correlated traits may drive the evolution of perennial ecotypes in this species. Total integrated selection consistently favored larger flowers in this perennial habitat, potentially explaining the observation that perennial populations have larger flowers than annuals throughout the species range . Flower size has been shown to be a target of selection in several annual populations of M. guttatus . However, the direction of selection on flower size is both spatially and temporally variable due to positive genetic correlations between flower size, flower time, and ovule and pollen production per flower . Thus, in an annual habitat, large flowers decrease survival to reproduction by delaying flowering but increase seed set per flower, and the net pattern of selection on flower size depends on the timing of drought-induced mortality . Although I did not detect correlations between flower size and flowering time directly, flower size was frequently positively correlated with flowering stem diameter, which in turn was often correlated with later flowering. Further, selection on flower size only occurred through components of fecundity. This suggests that the longer growth season in perennial habitats ameliorates viability selection on flower size, resulting in strong fecundity selection for larger flowers.

For multivoltine insects, longer growing seasons can also increase the number of generations completed per year, leading to population growth that might facilitate range expansion if host plants are available. Most predictions on plant species’ range shifts are predicated on the assumption that abiotic factors determine range edges; however, biotic factors can also contribute to range limits . There is also growing evidence that biotic factors, such as herbivores and disease can interact with abiotic factors to determine the trailing range edges of some plant species. However, the factors that drive range limits at leading and trailing edges remain unknown for most species. Biological collections typically have associated metadata describing when and where collections were made, and therefore provide rich data on species distributions and distributional shifts over time. Species distribution models are commonly used to map past and present distributions, but they are intrinsically limited by the number and representation of input records, and, in the case of global change research, the number of records available from before and after global change. The extensive digitization efforts currently underway for insect and plant specimens will improve our predictions and ability to track changing distributions. For well sampled plant species, we might also be able to investigate changes in herbivory at poleward range edges to determine if it has declined over time as plant ranges expand into novel habitats—an extension of the enemy release hypothesis associated with species invasions, discussed below. Larger digital collections of insect herbivores will provide the opportunity to compare range shifts across insect clades and to identify traits that govern range expansion and contraction. For example, we might expect that warmer winters will disrupt winter diapause for many insect species, container size for blueberries leading to range contraction and decline, while those that do not have diapause will benefit from higher rates of winter survival.

However, it is also possible that insects with diapause are more likely to maintain phenological synchrony with their hosts. We might then predict that plant –herbivore interactions would vary not only with warming temperatures, but also with the relative proportion of insect herbivores with winter diapause. Currently, widespread data on which insect species diapause is not available. Information deposited in centralized databases on diapause along with other life-history traits that might mediate insect herbivore range shifts would allow us to make better predictionson how herbivore damage will change over time. Species traits might also determine whether species shift over time or space, and how these two responses trade-off . The ability of insect herbivores to switch host plants may be another factor that constrains or facilitates herbivore range expansion, and thus plant –herbivore interaction strengths. Specialized insects that do not feed on newly encountered plant species may be limited in their geographical spread, whereas more generalist herbivores would be less constrained. Herbaria may capture such switches to novel hosts, showing up as new types of herbivore damage on specimens as host plants and their insect herbivores shift their distributions and provide the opportunity for novel plant –herbivore interactions. For example, leaf mines and galls—which are preserved on herbarium specimens—are made by a wide variety of insect herbivore taxa, including some of the most diverse groups of insects—Lepidoptera, Coleoptera , and Diptera —and are often specific to insect genera or species . The Lepidoptera that make leaf mines are not well represented in long-term citizen science data because leaf miners are typically micromoths, which are not the focus of long-term observations, and leaf mining and galling damage are only rarely included in herbivory studies, which tend to focus on chewing damage. Thus, herbarium specimens provide a record of a unique insect herbivore fauna not represented in long-term herbivore monitoring or herbivory studies.

Herbarium specimens may also provide data on a key hypothesis in global change biology that is based on theory which dates back to Darwin: the role of natural enemy release in species invasions. The enemy release hypothesis describes the escape from native predators and parasites when species are introduced into novel habitats. While there is evidence that introduced plants escape their native herbivores, it is unclear how long this ‘release’ persists. Herbarium specimens can provide rare long-term data on herbivory and disease pressure that allows us to resolve this question. In a well-documented example, Schilthuizen et al. used herbarium specimens to show that the non-native cherry tree, Prunus serotina, acquired higher rates of herbivory over time after its introduction to Europe, while its native congener, Prunus padus, had stable herbivory levels over the same time period. This led to field investigations into the contemporary herbivore communities for these congeners, which revealed that, surprisingly, P. serotina had a richer herbivore community than the native P. padus, and that P. serotina had acquired specialized herbivores from other native host genera. This supports the hypothesis that non-native plants accumulate herbivore taxa over time in their novel habitats, which might have significant implications for plants that shift in their geographical distributions.Urbanization affects insect herbivores via a variety of mechanisms, including habitat fragmentation, habitat and host plant loss, and introduction of novel host plants that attract and support non-native herbivore communities. Given these concurrent pressures, the effects of urbanization on plant – herbivore relationships are complex and varied . However, in recent years, it has become increasingly clear that a key aspect of urbanization, the urban heat island effect, can drive relationships between plants and herbivores and may uniquely inform climate change predictions.

The urban heat-island effect—the local warming of urban areas relative to surrounding countryside—increases urban temperatures 1– 128C higher than rural temperatures. Thus, local warming caused by urban development is similar in magnitude to warming expected globally over the next 100þ years, and it has therefore been suggested that cities may provide insights into the future effects of climate change. Like global warming, urban warming drives phenological advance in plants and insect herbivores. For example, plants leaf out and flower earlier in cities than in nearby rural areas, and urban heat is associated with earlier egg production for certain insect herbivore species. While the effects of warming global temperatures on the synchrony of plant –herbivore interactions is still generally unresolved owing to a lack of data, these relationships can be studied across urban temperature gradients, and there is some evidence for reduced synchrony between insect herbivores and their natural enemies as a result of urban warming. Because of the parallels between the abiotic and biotic effects of urban and global warming, natural history collections from urban areas may allow us to more broadly predict how global climate warming will affect interactions between plants and their insect herbivores. Phenology data from specimens—e.g. flowering, leaf-out, insect flight— paired with data on urbanization intensity in the areas where specimens were collected could inform predictionson phenological change and synchrony for a broad range of plant and herbivore species. Specimen data from urban areas are, perhaps surprisingly, plentiful. A recent study shows that across three areas with large digitized herbarium collections—the US, South Africa and Australia—plant specimens are often collected close to natural history museums or roads. Thus, specimens could be used to explore urbannatural gradients. Like temperature, urbanization can be easily assigned to historical specimens via contemporary measurements or existing data. As a proxy for urbanization, we can use human population density data from censuses, which many countries have been collecting since the early 1900s, and in some places urbanization can be translated from historical maps or as impervious surface derived from satellite imagery. One novel approach might be to derive markers of urbanization from the herbarium specimens themselves, for example, signature pollutants , although disentangling the contributions of different drivers would then present additional challenges. In recent years, raspberry grow in pots growing evidence shows that urban warming may increase abundance of certain herbivores, notably sapfeeders, potentially leading to more insect damage on urban than on rural plants, a pattern that has been documented by entomologists for over a century.

Sapfeeding herbivores, such as scale insects and aphids, are often preserved on leaves and branches and thus may provide insights into changing herbivore pressure in response to urbanization. In a recent study, Youngsteadt et al. counted armoured scale insects on branches of herbarium specimens of red maple Acer rubrum and on branches of live trees across an urban warming gradient. Using these data, they showed that interannual warming and urban warming may have surprisingly congruent effects on scale insect prevalence. In box 2, we discuss how herbarium specimens might be used to investigate more complicated interactions between multiple trophic levels, relationships that could inform biological control efforts and management of urban plants. While the urban heat island effect benefits certain herbivores that survive within the urban matrix by advancing their phenology and increasing their abundances, urbanization also excludes some insect species—a pattern which has been documented with insect museum specimens—making the effects of urbanization on herbivore damage to plants difficult to predict. Long-term records of butterfly flight from Britain showed that habitat loss is associated with butterfly decline, especially for species that are less mobile and are habitat specialists. Relatedly, a recent study across 16 European cities showed that leaf chewing damage was lower in cities relative to nearby rural areas, perhaps driven in part by higher rates of bird and ant predation on insect herbivores in cities than in rural areas. Thus, a pattern that might be emerging from the literature is that certain sap-feeding insects benefit from urban heat , while leaf chewing and the insects that cause this type of damage, notably Lepidoptera, decline in response to loss of habitat and host plants caused by urbanization. This finding suggests that the effects of bottom-up versus top-down forces driving insect herbivore fitness might differ among feeding guilds . Measurements of broad-scale chewing herbivory , presence of sap-feeders, and incidence of sooty mould as a proxy from herbarium specimens, along with insect herbivore occurrence data, could be used to test this hypothesis . In addition to describing the effects of urbanization at the local scale, museum specimens may also reveal how urbanization affects species distributions at broader spatial scales. For example, while urbanization may disrupt poleward range expansion for some species, it is possible that cities serve as warm habitat stepping stones for species with long-distance dispersal mechanisms, facilitating their poleward expansion. The insects that create leaf mines have been described as ‘aerial plankton’ because they tend to disperse long distances. Herbarium specimens might capture this rapid northern expansion of leaf mining insects and provide a record of shifting interactions with native plants that may be more likely to respond in time than space —see box 1. Similarly, a comparison of herbivory damage on herbarium specimens of non-native plants in urban versus rural environments might provide insights into one pathway towards species invasion. Many non-native plant species are introduced into urban areas, and urbanization may provide warm, enemy-free space where they can establish and subsequently expand. Though the role of urbanization in natural enemy release and subsequent invasions is not well characterized, we might predict that non-native plants escape their natural enemies in urban areas and experience increased herbivory rates when they move into natural areas where they encounter a higher diversity of herbivores.Responses to global change, such as those in space and time, discussed above, encompass plasticity in behaviour or physiology and distributional shifts, which may be rapid. However, there is growing evidence that evolutionary responses might also be rapid, assuming there is sufficient standing genetic variation for selection to act upon. While fluctuating selection can maintain this standing variation, providing the raw material for future adaptive responses, strong directional selection, such as that imposed by anthropogenic climate warming, can erode genetic variation and potentially impede evolutionary adaptation, elevating population extinction risk . Insect herbivores and the plants they feed upon are locked in an evolutionary arms race, and insect herbivory drives contemporary plant evolution, changing plant allele frequencies within a few generations. It is likely that climate-induced shifts in herbivory will impose additional selection on both plants and insect herbivores already under pressure from direct effects of climate change. Natural history collections that span multiple generations can provide a record of evolutionary changes and constraints. It can be difficult, however, to disentangle plastic and evolutionary responses.

After hand pollination, we re-bagged flowers with breathable mesh bags to exclude pollinators while simultaneously permitting the flower to experience conditions like those experienced by flowers open to bees . After the bee-pollinated and hand-pollinated flowers closed , we randomly selected flowers to estimate either pollen deposition, or fruit and seed set. To estimate pollen deposition, we first used a single-edged razor blade to remove bee and hand-pollinated floral stigmas, which we immediately placed in a solution of 70% ethanol and basic fuchsin dye . We randomly selected two stigmatic lobes , counted all pollen grains present on these lobes under a dissecting microscope at 50x magnification, and then calculated the mean pollen count between both lobes . We calculated the total stigmatic pollen count as the mean pollen deposited per lobe plus any stained grains present in the ethanol solution. As expected, pollen deposition was higher in hand-pollinated plants: total pollen count on stigmas pollinated by hand was more than two-fold higher compared to that on stigmas pollinated by bees . We harvested fruit 50 days after pollination . Developed seeds from mature fruit were dried and weighed. We report total seed mass per fruit per plant as a measure of seed set. Mean seed mass did not depend on seed number in this experiment . For all statistical analyses performed in this study, we used R version 3.6.1 and used the package ggplot2 to prepare figures . We inspected qq plots to test for normality and used Bartlett tests to assess homogeneity of variances. We also tested the residuals of each model with the Shapiro-Wilk test for normality.

For bee-pollinated plants in the Temperature x irrigation experiment, we used general linear models with two fixed factors, soil moisture and temperature , raspberry plant pot to examine their direct effects on individual floral traits : total flower number, self and non-self flower number, flower size, nectar volume, nectar concentration, pollen mass, and pollen viability. We also used general linear models with soil moisture and temperature as fixed factors to examine bee visitation and behavior rates in flowers. We conducted a second set of analyses with an additional fixed factor to test whether or not differences in fruit set and seed set resulting from soil moisture and temperature variation could be mediated by interactions with pollinators. For all analyses in the Temperature x irrigation experiment, plant is the experimental unit of analysis. All plants were given at least one chance to set fruit from a pollinated female flower. For plants that set more than one fruit , we used mean values for total seed mass.For the floral trait analyses, the response variables of total female flowers and total available self male flowers were right-skewed and thus log10 transformed to improve normality of the residuals. Pollen mass in male flowers was right-skewed and thus square-root transformed to improve normality, although pollen mass still did not have equal variances after transformation. Nectar concentration in female flowers and proportion viable pollen in male flowers were left-skewed and thus square transformed to improve normality, although pollen viability still did not have equal variances after transformation. Variances were marginally equal for nectar volume in male flowers. For the bee visitation analyses, all rates of bee visits per flower per minute were proportions and thus arcsine square root transformed to improve normality; however, except for Apis visit rate to female flowers, the residuals were still not normal even after transformation.

For the bee behavior analyses, all behavior rates were right-skewed and thus log10 transformed to improve normality of the residuals; however, the residuals were still not normal for Eucera behaviors in male flowers and the variances were still not equal for Apis pollen collection per min. For the fruit and seed set analyses, the residuals were not normal for fruit set. To determine if bee-pollinated plants experienced pollen limitation, we calculated pollen limitation using the pollen limitation index, L = 1 – . In this equation, B represents total seed mass for a bee-pollinated plant, and H equals total seed mass for a hand-pollinated plant. For each hand-pollinated plant in the equation, we matched a corresponding bee plant that had similar mean plant soil moisture and was from within the same experimental group. For hand and bee plants with more than one fruit in July, we used mean values of total seed mass for each plant. We used a general linear model containing soil moisture and temperature to examine their direct effects on pollen limitation. Soil moisture values used in the analysis are those of the matched bee-pollinated plants. We also estimated pollen limitation using a second method in which we used all bee-pollinated plants and the mean values of their matched hand-pollinated plant treatment groups. This second method yielded results that are qualitatively similar .From June through September 2017, we tested how irrigation level affected the degree to which bee pollinators of squash transfer self pollen versus non-self pollen. This particular research focus was stimulated by one of the results of the Temperature x irrigation experiment, namely that the availability of non-self, male squash flowers increased with increasing soil moisture experienced by individual plants . Given this result, pollinators may be transferring increasing amounts of self pollen with decreasing soil moisture, which may cause reduced seed set.

In Irrigation experiment I we grew squash in a similar manner to that in the Temperature x irrigation experiment, but in the present experiment we grew C. pepo plants under different levels of irrigation and did not manipulate temperature. We used a drip-line system to irrigate plants every morning; the irrigation treatment included two levels: 1.3L water/plant/day and 0.38 L water/plant/day . We monitored soil moisture levels as in the Temperature x irrigation experiment and found that volumetric water content decreased by an average of 17% for plants in the low irrigation treatment . Soil moisture levels again exhibited substantial variation within each experimental group. Mean volumetric water content, for example, varied from 38 – 63% to 10 – 54% . For this reason, we again consider soil moisture as a continuous variable in all statistical analyses but use treatment group designations in the organization of the experiment. All analyses are restricted to data collected from July 28 – August 25 during the height of flowering and pollinator visitation. To test for differences in the transfer of self pollen and non-self pollen as a function of irrigation level, we used florescent powdered pigments to track bee movements among plants . On each day of the experiment, we identified 1-2 focal plants that each had one female flower and at least one male flower open that day. Before allowing bees access to flowers, we used flat toothpicks to apply powdered DayGlo© fluorescent pigments to the anthers of focal male flowers . On a given day, the male flowers of each focal plant received its own unique pigment color; all male flowers on non-focal plants received a different pigment color. Each day we switched the pigment color assignments between focal and non-focal plants to mitigate for any color preferences exhibited by bees. When bees contacted the anthers of a male flower with pigment, blueberry production pollen grains as well as pigment particles adhered to the bees’ bodies. Therefore, the number of pigment particles acted as a proxy for the number of pollen grains transported by bees to the stigmas of the female flowers . We estimated pollen deposition as follows. Following pollination, removed stigmas were immediately placed in 100% ethanol . Prior to adding basic fuchsin solution to dye pollen grains, we used a dissecting microscope to count pigment particles under 40x magnification . We then added basic fuchsin solution and used a dissecting microscope to count the number of pollen grains on the entire stigma and in the ethanol solution containing the stigma .

Mean total pigment particle count on bee-pollinated stigmas was highly correlated with mean total pollen grain deposition . To assess whether or not irrigation level affects the deposition by bees of self versus non-self pollen, we used general linear models to compare pigment and pollen deposition as a function of soil moisture.From June through September 2018, we conducted a field experiment that involved irrigation and hand-pollination to test how pollen source , and pollen identity affect the seed set of plants grown under different levels of soil moisture . To hand pollinate female squash flowers, we used identical methods as those employed in the Temperature x irrigation experiment, except that we reduced the amount of pollen deposited to stigmas to emulate levels observed in bee-pollinated flowers, and to decrease pollen competition below levels that likely occurred in the hand-pollination treatment in the Temperature x irrigation experiment. When stigmatic pollen deposition is adjusted to levels comparable to those delivered by bees, reduced pollen competition would presumably allow the majority of pollen grains to germinate and not just those that were most viable . This scenario should thus provide greater sensitivity to detect differences in how plants respond to self or non-self pollen under varying degrees of water stress. Squash rearing was performed in a similar manner to that in the Temperature x irrigation experiment, but in this experiment we grew C. pepo plants under different levels of irrigation and did not manipulate temperature. We used a drip-line system to irrigate plants every morning; the irrigation treatment included two levels: 2.4 L water/plant/day and 0.38 L water/plant/day . Mean volumetric water content for plants in the low irrigation treatment was 11% lower than that of plants in the high irrigation treatment . The volumetric water content for plants in the high irrigation treatment ranged from 41 – 54%, whereas that of plants in the low irrigation treatment ranged from 31 – 41%. All analyses are restricted to data collected from August 3 – September 8 during the height of flowering and pollinator visitation. Once squash plants began to flower, we hand-pollinated female flowers with either self pollen or non-self pollen from plants within the same irrigation group. We measured pollen deposition as in Irrigation experiment I and fruit set and seed set as in the Temperature x irrigation experiment. To test how soil moisture affects the seed set of flowers pollinated with pollen that varied in terms of its source and its identity, we considered two different measures of soil moisture. First, we used soil moisture values of the plants that produced focal female flowers. Second, we used the mean soil moisture experienced by the plants that provided pollen used for hand pollination . In the fruit set analysis, we did not use mean soil moisture experienced by the plants donating pollen because some plants had multiple chances to produce non-self fruits, and each of these chances utilized pollen from different plants with different soil moistures. To test how flowers pollinated with self pollen versus non-self pollen responded to soil moisture variation in terms of fruit set, we ran separate general linear models , each containing soil moisture as a fixed factor. Given the number of zero values in our data set , we used a zero-inflated negative binomial model using package gamlss to analyze seed set, which included zero values. This model contained two fixed factors, soil moisture and pollen type , and one random factor . We adopted this approach because as mentioned previously, plants in this experiment were given multiple opportunities to set fruit, and plants had the opportunity to grow both self and non-self fruits. We ran these models first using the plant’s soil moisture and then again using the mean soil moisture of the two plants donating pollen for the non-self fruits.From June through September 2018, we conducted a field experiment that involved irrigation and bee-pollination to test how pollen source affected the seed set of plants grown under different levels of soil moisture. For this experiment, we allowed bee visitation on plants grown under a gradient of soil moistures. Therefore, bees could move freely between flowers in both high-irrigation and low irrigation treatment groups and, consequently, deposit pollen from both high and low moisture plants on stigmas of plants grown under different levels of soil moisture. Squash rearing was performed in a similar manner to that in the Temperature x irrigation experiment, but in this experiment we grew C. pepo plants under different levels of irrigation and did not manipulate temperature.

Flavonols play an important role as antioxidants; for example, they protect ascorbic acid from autoxidation in juices and which can lead to juice discoloration. Although flavonoids are abundant in fruit, and fruits or beverages can be a significant source of dietary flavonoids, levels will vary depending on the varieties, environmental conditions, soil, and climatic factors. Berries are a good source of quercetin and its derivatives , whereas the most abundant dietary flavanone glycoside is hesperetin-7-O-rutinoside present in citrus fruits. Peterson et al. reported that the most prevalent dietary flavanone aglycones are naringenin, hesperetin, isosakuranetin, and eriodictyol. ,e same authors demonstrate that a citrus fruit is also a primary source of narirutin, eriocitrin, didymin, neohesperidin, naringin, hesperidin, neoeriocitrin, and poncirin. ,e ratio of these compounds to each other can vary. For example, narirutin and naringin were detected in grapefruit in high ratios, while the levels of hesperidin and narirutin in oranges and eriocitrin in lemons were even higher. In addition, some flavanone glycosides such as 7-rutinoside are tasteless, in contrast to neohesperidin , naringin, and hesperetin which have an intense bitter taste isolated from bitter oranges and grapefruit. Apigenin is another key flavone found in fruits, vegetables, spices, and herbs and is abundant in grapefruit, beverages, some vegetables, and herbal plants such as chamomile. Isoflavones are present in plants in the glycosylated forms but are converted to aglycone forms through the action of intestinal microflora. Isoflavones are detected commonly in legumes such as green beans, fava, and soybeans, and among them, container size for raspberries genistein -4H-1-benzopyran-4- one and daidzein -4H-1- benzopyran-4-one are the two major forms of dietary isoflavones and are consumed in soy products.

Fermented soy products also contain an additional seven isoflavone aglycones in significant levels. Due to the structural similarities to human hormone estrogen, isoflavones have potent estrogenic properties. Anthocyanins are another important class of flanovids that are colorful water-soluble glycosides and acylglycosides of anthocyanidins. 3-O-glycosides or 3,5-di-O-glycosides of malvidin, delphinidin, pelargonidin, cyanidin, petunidin, and peonidin are known as the most common natural anthocyanins and are classified based on the number and position of hydroxyl and methoxy groups. Anthocyanins are responsible for the brilliant colors of various plant parts including flowers and leaves and especially fruits having red, blue, purple colors, particularly strawberries, blueberries, black currants, cherries, raspberries, and red and purple grapes. Anthocyanidins are also responsible for the color of red wines. ,eir color based upon the degree of methylation and with pH is discrete from other phenolics by the range of colors each forms. Color differences of anthocyanins depend on the substitutions of the B ring, the pattern of glycosylation, and the degree and nature of esterification of the sugars with aliphatic or aromatic acids, and also on the pH, temperature, type of solvent, and the presence of copigments. Berries are a good source of anthocyanins, and 100 g of berries can provide up to 500 mg of anthocyanins. Flavan-3-ols have been previously reported as an antioxidant, chemopreventive, and immunoregulation agents. Procyanidins exist in a wide range of foods and often exist in foods in a range of galloylated forms. Most widely used techniques for phenolics are HPLC , LC/MS, GC , GC/MS, UV-Vis spectrophotometry, mass spectroscopy, electrochemical, and fluorometric methods. Liquid chromatography mass spectrometry is used to determine phenolics in both APCI and ESI techniques, ABTS+ and DPPH.

Sample preparation and extraction methods varied widely based on the nature of the sample matrix of the fruit or vegetable and based on the chemical structures of the phenolic compounds being extracted. As most samples contain a mixture of simple and complex polyphenolic compounds, such as phenolic acids, flavonoids, anthocyanins, and proanthocyanins, it is critical to choose a suitable method for sample preparation and extraction. Proteins, carbohydrates, lipids, or other elements may play negative effect to extraction of phenolics. In addition, it is not always possible to extract fresh samples, and special preparation techniques such as lyophilization, nitrogen pulverization, or drying may be needed. Particle size of extracted material and solvent-to-solute ratios need to be considered. As seen in Figure 1, there are many reliable qualitative and quantitative methods available for the measurement and characterization of the phenolic content in different natural products. Moreover, the success of these techniques will depend on the most effective sample preparation and extraction methods. Extraction efficiency is greatly influenced by solvent choice and composition and plays a critical role in the extraction yield of phenolics from fruits and vegetables. Generally, for the extraction of phenolics, water, acetone, ethyl acetate, alcohols , and their various percentages of mixtures are used. In addition to the solvent type extraction conditions, parameters such as temperature and duration also influence the yield of phenolics.Khoddami et al. previously reported that recovery of phenolics varied from one sample to another sample. It is also reported that acid- or base-catalyzed hydrolysis is also an important consideration for the stability of the phenolics in extracts. Davidov-Pardo and Marn-Arroyo reported that the extraction pH plays an important role in the extraction efficiency of phenolic compounds, and the same authors implied that catechins and their isomers are detected more efficiently in alkaline conditions as compared with acidic ones. Extraction of phenolic compounds are commonly done using either liquid-liquid or solid-liquid extraction technique. However, liquid-liquid extraction has some disadvantages because of using costly and potentially toxic solvents. For this reason, improved extraction methods such as solid-phase microextraction and solid-phase extraction techniques are used to extract phenolics from liquid samples.

In general, inexpensive and simple methods such as soxhlet, reflux, and maceration processes are the more conventional procedures used to recover phenolics from solid samples. In addition, ultrasound-assisted extraction , microwave-assisted extraction , ultrasound microwave-assisted extraction , supercritical fluid extraction , subcritical water extraction , and high hydrostatic pressure processing are the methods that help us to shorten extraction times and decrease the release of toxic pollutants through reducing organic solvent consumption and are relatively simple to perform. Pulsed electric field is also another extraction technique that can be applied at room temperature conditions and performed in a matter of seconds requiring low energy to increase cell membrane breakdown in mass transfer which were applied previously in several fruits such as strawberry and grapes.,e analysis of phenolic acids and flavonoids by liquid and/or gas chromatography techniques is the most widely and commonly applied methods for the quantification of phenolics in fruits and vegetables. In addition, spectrophotometric assays are used as nonspecific methods used for evaluating the levels of phenolics in many fruits and vegetables.Although, fruits differ in the quantity and types of phenolic antioxidants, degree of conjugation, and composition of sugar, total phenolic compounds can be estimated in fruits using the reagent proposed by Otto Folin and Vintila Ciocalteu and recently modified by Li et al.. ,is Folin–Ciocalteu method is robust, highly reproducible , convenient, and fast, requiring only a UV spectrophotometer. ,e method is typically standardized with either gallic acid, rutin, or a combination of pinocembrin/galangin. ,e Method is based on a reaction of the chemical reagent with phenolic electron transfer. ,e phenolic compounds are oxidized to phenolates by the reagent at alkaline pH in a saturated solution of sodium carbonate resulting in a blue molybdenum-tungsten complex and can be measured at 765 nm. ,e absorbance of each sample can be compared with those obtained from the standard curve, and the obtained data are expressed as µmol gallic acid equivalents per gram of fresh or dry matter. Because the reaction is quantitative and presumable, the analysis of a mixture of phenols can be recalculated based on any other standard. ,e assay comprises of monophenols and provides predictable reactions based on the phenols and provides measuring of all compounds readily oxidizable under the reaction conditions.Recently, chromatographic techniques such as highperformance liquid chromatography , HPLC electrospray ionization mass spectrometry , big plastic pots gaschromatography-mass spectrometry , capillary electrophoresis , and near-infrared spectroscopy techniques are developed for identification, separation, and quantification of phenolics. Phenolic content of plant materials can be measured and identified using HPLC employing different stationary phase-solvent combinations and various detectors. HPLC relies on comparisons of unknown compounds with standard reference compounds to make both qualitative and quantitative analytical measurements. Columns can be selected to impart specific separations based on the stationary phase type and the size and structure of the packing materials to which the stationary phase is bound to [52].

Detector choice can also be manipulated to enhance detection and especially quantifi- cation. Phenolic compounds can easily be measured using UV-Vis, photodiode array detection , fluorometric detection , and electrochemical detection . Each stationary phase-detector combination will provide specific information on the phenolic composition of a sample. For example, UV detection can be used to measure benzoic acid at 246–262 nm, gallic acid at 271 nm, and 275 nm for syringic acid. Two different wavelengths 225–235 nm and 290– 330 nm can be used to measure cinnamic acids, but the common wavelength of 280 nm is issued for the general analysis of phenolics. However, many factors such as sample purification, column and detector types, solvents used as mobile phase and solvent purity, and their pH affect HPLC analysis of phenolics. It is previously reported that mixtures of water, methanol, acetonitrile, formic and acetic acids, and trifluoroacetic acid are used for mobile phase for phenolic compounds in reversed phase chromatography using octadecyl silica columns. Generally, among the HPLC detectors, UV-Vis and DAD detectors are more common compared to the fluorometric detection . Common stationary phases include C18 RP columns employing an acidified mobile phase and ammonium acetate buffers of organic solvents . Detection efficiency can be improved by using SPE cartridges composed of styrenedivinylbenzene to purify phenolic compounds from crude extracts prior to HPLC analysis. ,e wavelength selected for monitoring phenolics is an important criterion and generally ranges between 190 and 380 nm. Gradient elution is generally preferred rather than isocratic elution. Some of the authors previously reported that phenolics such as flavonones, flavonoids, and flavan-3-ols of plum, blueberry, raspberry, strawberry, orange, apple, and tea are possible to be measured by common HPLC techniques. In general, for identification and quantification of phenolics, individual stock solutions of each standard are prepared in methanol and stored at −20°C until analysis. ,e working standard mixture solutions are made by diluting the appropriate amount of each stock standard solution to obtain at least 5 calibration levels. Measurements of flavanols, hydroxycinnamates, flavonols, and anthocyanins of fruits can be detected at 280, 320, 360, and 520 nm by using HPLC. External standards are used to quantify the phenolic compounds. Stable isotopes can also be used to quantify phenolic compounds when HPLC-ESI/MS is being used as described below.HPLC-ESI/MS is used to increase the range of phenolic compounds detected in a sample and to improve sensitivity as compared with standard chromatographic methods. HPLC-ESI/MS is a robust and selective quantification method that is effective at measuring the complex array of phenolics typically found in fruits and vegetables. Mass spectrometry methods can be performed on a variety of instruments including electrospray ionization ion trap instruments, triple quadrupole instruments , and time-of flight instruments . ,e mass spectrometer is an analytical detector that gives both qualitative and quantitative measurements based on separation of ions by their m/z ratio and 0.01% correction. Mass spectrometry involved three stages: ionization, mass analysis, and detection of ions. Separation of phenolic compounds is best achieved in aqueous-organic extracts of foods with HPLC prior to MS analysis although GC can also be used. ,e most common solvent reduction and ionization technique is electrospray ionization . ,is can be performed using different voltages to create negative or positive pseudomolecular ions that can be accelerated into the mass analyzer. ,e mass analyzer separates ions based on the flight path as with a magnetic /electric field separation, time-of-flight in a filed free region, or by altering ion trajectories using quadrupole and ion trap mass analyzers. Detection is usually achieved with an ion multiplier tube. Triple quadrupole analyzers and ion trap analyzers are often used when higher sensitivity and specificity, or structural information is required for identification. Fidelity of MS measurements can be increased using MS/MS techniques. For example, a common technique is to create product ions through collision-activated dissociation of selected precursor ions in the collision cell of the triple quadrupole mass spectrometer , analyzing the fragment ions in the second analyzer of the instrument .

Despite being non-native in the great majority of places where they were studied, honey bees were as efficient, on average, as the native floral visitors studied. The fact that honey bees are as efficient as the average pollinator, even where non-native, is perhaps not surprising since they are a super-generalist pollinator. As super-generalists, the honey bee is adept at extracting pollen and/or nectar from many plant species within a landscape rather than being specialized on one or two plant species. Such a generalist will develop strategies to exploit many types of floral architectures, although efficient exploitation of a floral resource does not necessarily correspond to efficient pollination. Body size may help to explain why honey bees are relatively efficient pollinators of the majority of plants they visit, despite their floral diversity. Honey bees have fairly large bodies, which may better facilitate pollen transfer compared to small bodied pollinators; for example in commercial apple, larger body size of bumblebees was suggested to explain their higher pollen deposition rates relative to smaller pollinator taxa . Honey bees were however less efficient than the top pollinator. This pattern may be partially a statistical artifact. Whenever multiple floral visitors are studied, even if they are all in fact equally efficient, the estimate for the efficiency of honey bees is expected to be less than that of the top pollinator /n of the time where n is the number of pollinating taxa studied. However, blueberries in containers growing honey bees were significantly less efficient than the top pollinator measured in 15 of the 34 plants studied. Honey bee generalist pollinating behavior may make them as proficient as the average floral visitor. However their generalist strategy could also explain the gap in efficiency compared to the top pollinator, which may be more specialized, at least in some cases.

Although honey bees were less efficient than the top non-honey bee pollinator, honey bees were no less important. Lack of a statistical difference between pollinator types may partly be due to lower sample size. However, given the data at hand any lack of per-visit performance by honey bees in comparison to the top non-honey bee pollinator was made up for by relative visitation frequency. We conclude that for plant species where honey bees are the most frequent floral visitor, they may often account for the majority of pollination services. Honey bee relative efficiency did not depend on whether or not a plant was domesticated, again suggesting that bees are reasonably efficient across species from a wide range of plant families and floral architectures. However, unlike undomesticated plants, honey bees were found to be less important for agricultural plants than the other pollinators studied though the sample size for domesticated plants is quite small . These plants may have been selected for study partially because of high visitation rates of non-honey bee species. Low relative honey bee importance in this small group of agricultural crops may also be due to special pollination systems for plant species studied; for example, tomato is buzz pollinated, but honey bees don’t perform buzz pollination and species of Cucurbita are visited by specialist bee species from the genera Peponapis and Xenoglossa that become locally abundant and move between flowers much more rapidly than honey bees, leading to high visitation rates. In sum, for plants where honey bees are frequent floral visitors, we can generally expect them to provide adequate pollination services in natural communities. As a result of habitat fragmentation or climate change, the pollination services from specialist pollinators species may diminish or be lost . Where specialist pollinators have been lost, our results suggest that honey bees may be able to substitute for the pollination services formerly provided by the pool of diverse pollinators originally present, for the plants they visit.

However, there may still be cases where, for particular plant species, the switch from one or more native pollinators to predominant visitation by honey bees could cause reproductive declines. Furthermore, given that, honey bees do not visit all plant species within natural communities , the integrity of plant reproduction on an ecosystem scale may still suffer with pollinator diversity loss, even where honey bees increase in abundance. Therefore, while honey bees, even where introduced, can provide important pollination services to naturally occurring plants, maintenance of a diverse pollinator assemblage may still be required to ensure adequate reproduction of entire plant communities. Further research is needed in order to more thoroughly understand community wide impact of changes to pollinator assemblages in response to current and future environmental stressors. Chapter 1, in part is currently being prepared for submission for publication of the material. Hung, Keng-Lou James; Kingston, Jennifer M.; Albrecht, Matthias; Holway, David A.; Kohn, Joshua R. Keng-Lou James Hung was the primary investigator and author for this paper.Approximately one-third of food produced globally is lost or wasted, yet fewer resources are devoted to postharvest research and development than to efforts for improving productivity. The modular design of plants allows plant tissues and organs to remain biologically active even after harvest. Therefore, capitalizing on the ability of harvested vegetables and fruits to continue to sense and respond to diverse stimuli, similarly to intact plants, may be a powerful approach to promote postharvest quality. Research demonstrating the biological advantage of a functional circadian clock in plants led us to investigate whether maintaining diurnal cycles may promote longevity and therefore reduced yield loss during postharvest storage of vegetables. The circadian clock enables plants to anticipate and prepare for the daily environmental changes that occur as a consequence of the rotation of the earth. Coordination of plant circadian rhythms with the external environment provides growth and reproductive advantages to plants, as well as enhanced resistance to insects and pathogens. The circadian clock also regulates aspects of plant biology that may have human health impact, such as levels of carbohydrates, ascorbic acid, chlorophyll, and glucosinolates in edible plant species.

Plants exhibit exquisite sensitivity to light stimuli, and isolated plant leaves maintain responsiveness to light after harvest and can continue light-dependent biological processes, such as photosynthesis. Additionally, the clocks of postharvest fruit and vegetable tissues can been trained with 12-hour light/12-hour darkness cycles producing rhythmic behaviors not observed in tissues stored in constant light or constant dark. A few studies have examined the effects of light on performance and longevity during postharvest storage. For example, light exposure delays broccoli senescence and yellowing but accelerates browning in cauliflower, a close relative of broccoli . Other studies report that light exposure to broccoli during postharvest storage either provides no additional benefits or decreases performance. Postharvest light exposure improves chlorophyll content in cabbage, but leads to increased browning of romaine lettuce leaves. Although exposure of spinach to light during postharvest storage can improve nutritional value, light can also accelerate spinach water loss, leading to wilting. Together, these findings are inconclusive as to whether light exposure during postharvest storage can be generally beneficial, and the variation of the results may be attributable to differences in the plant species examined and the specific conditions used during postharvest storage, such as lighting intensities, temperature, humidity or packaging. Alternatively, light may be advantageous but only if present in its natural context with 24-hour periodicity because of such timing on circadian clock function. This study aimed to examine whether mimicking aspects of the natural environment predicted to maintain circadian biological rhythms during postharvest storage of green leafy vegetables improves performance and longevity compared to postharvest storage under constant light or constant darkness. We focused this work on several popular and nutritionally valuable species, planting blueberries in containers including kale and cabbage , members of the Brassicaceae family with worldwide production of approximately 70 million tons. In addition, we analyzed green leaf lettuce and spinach , which have worldwide production of approximately 25 and 22 million tons, respectively. Here, we report on the promotion of postharvest longevity, including tissue integrity and nutritional value, of green leafy vegetables by provision of 24-hour light/dark cycles during storage compared to storage under constant light or constant darkness.Fruits and vegetables after harvest can respond to repeated cycles of 12-hour light/12-hour dark, resulting in circadian clock function and rhythmic behaviors. Because a functional plant circadian clock is physiologically advantageous we sought to address whether postharvest storage under conditions that simulate day/night cycles, thereby potentially maintaining biological rhythms, would affect postharvest longevity. We chose to address this question using green leafy vegetables, including commonly consumed kale , cabbage , green leaf lettuce and spinach , because we anticipated that the leaf organ would likely maintain light sensitivity and responsiveness even after harvest. To begin to determine whether daily light/dark cycles during postharvest storage affects leaf longevity, we compared the overall appearance of leaf disks that were stored at 22°C under cycles of 12-hour light/12-hour darkness versus leaf disks stored under constant light or constant darkness for various lengths of time . Under cycles of 12-hour light/12-hour darkness, kale leaf disks were dark green after 3 days of storage . After 6 days and 15 days of storage, the kale disks showed lighter green coloration than the kale disks stored for 3 days . However, the kale leaf disks stored under constant light were lighter green than the kale disks stored under light/dark cycles and showed some brown or yellow discoloration after 3 and 6 days . By 15 days, the kale leaf disks stored under constant light lost nearly all green coloration and showed light and dark shades of browning with shape changes resulting from leaf folding and shrinkage . The kale leaf disks stored under constant darkness resembled those stored under constant light, except that the 3-day kale samples were darker green than the 3-day constant light-stored kale leaf disks , suggesting that the constant light may have constituted a greater stress on the kale leaves than constant darkness. These results indicate that postharvest storage with daily cycling of light and darkness improved the appearance of the kale leaf tissue compared to storage under either constant light or constant darkness. However, the preservation benefit obtained from postharvest storage under light/dark cycles at 22°C appeared to be less than that provided by refrigeration; kale leaf disks stored at 4°C with constant darkness, were comparable in their dark green coloration whether stored for 3, 6 or 15 days . Cabbage leaf disks stored under cycles of 12-hour light/ 12-hour darkness showed brown spots along the disk edges that increased in intensity over the storage period of 7, 14, and 21 days . However, although the 7-day cabbage leaf samples were light green in coloration, the 14- and 21-day cabbage leaf disks stored under light/dark cycles had darker green coloration , suggesting increased photosynthetic activity over storage time. In contrast, although the cabbage leaf disks stored under constant light were also light green after 7 days of storage, the 14- and 21-day cabbage leaf disks were more yellow and included more brown discolorations . Remarkably, the absence of light exposure during post-harvest storage had a dramatic effect on the cabbage leaf disk coloration. Cabbage leaf disks stored under constant darkness at either 22°C or 4°C were pale tan or yellow after 3 days of storage . The constant darkness-exposed cabbage leaf disks stored at 22°C appeared nearly white in color by 14 and 21 days; those at 4°C had a yellowish appearance after 2 or 3 weeks of storage . Lettuce and spinach leaf disks tissue were nearly uniformly green, with little difference in color intensity between 3 and 6 days of storage under cycles of 12-hour light/12-hour darkness . By 9 days of storage under light/dark cycles, however, both lettuce and spinach leaf disks looked slightly less green, and most of the spinach leaf disks had distinct patches of yellow . In contrast, the loss of green coloration and increased yellowing over time was much more apparent in the lettuce and spinach leaf disks stored under constant light; the lettuce leaf disks were pale green by 9 days , and all the spinach disks had large yellow patches . After 6 and 9 days of storage under constant darkness, the lettuce disks had large wet patches of darkened tissue .

MS data was obtained using the positive scan mode for all of the extracts considered in the study. Compared to the underivatised UV-VIS and DPPH‚ chromatograms, the positive scan mode MS chromatograms show very different profiles . The cinnamon myrtle chromatograms are dominated by a peak that elutes with a retention time of 11 min. This peak does not appear in either the underivatised UV-VIS or DPPH‚ chromatograms. A small number of secondary peaks occur in both chromatograms, although they too do not appear to correspond to any major peak in either the underivatised UV-VIS or DPPH‚ chromatograms. Unlike the cinnamon myrtle, the negative scan MS chromatograms of the lemon myrtle are not dominated by a single peak, but show a number of peaks with similar intensity. Additionally, some of these peaks match in retention time to peaks that appear in the underivatised UV-VIS and DPPH‚ chromatograms. In particular, peaks with retention times of 3 and 4.5 min appear in a similar area of the chromatogram to peaks that respond to both underivatised UV-VIS and DPPH‚ . Additionally, the large peaks at around 12 min in the underivatised UV-VIS chromatograms appear as small peaks in the MS negative scan. As the number of peaks identified in the MS in positive scan mode was smaller than expected, growing raspberries in container the multiplexing experiment was repeated for the water extracts using both negative and positive MS scan modes. The water extracts were chosen as they showed the greatest number and intensity of antioxidant peaks in the DPPH‚ chromatograms. Both cinnamon myrtle and lemon myrtle showed a greater abundance of peaks in negative scan mode compared to positive scan mode.

Additionally, both chromatograms showed a large number of peaks eluting within the first 5 min of the chromatogram where the majority of the compounds that gave a response to DPPH‚ eluted. Table 1 shows the peaks that were detected in the DPPH‚ chromatograms along with the masses of those peaks as determined in the MS scans and possible identification based on the MS data. Due to the non-specificity of the MS scan, a number of the peaks that were identified in the MS data were due to more than one major m/z value indicating the presence of two co-eluting species being detected. Furthermore, a number of peaks that were detected in the DPPH‚ chromatograms did not show peaks in the MS data, indicating that these species did not ionise in the MS conditions used in the method. Additionally, it can be seen that a number of peaks that eluted had a m/z ratio that is either lower than typically observed in antioxidants, such as the peak at 0.5 min in the cinnamon myrtle water extract, or higher than that of typical antioxidants, such as the peaks at 3.3 and 4.6 min in the lemon myrtle water extract . This indicates that two different peaks may be eluting at these retention times, one of which is observed in the DPPH‚ chromatogram and the other that is observed in the MS chromatogram. Finally, it can be seen that where MS peaks were evident in both positive and negative scan modes, most of the peaks observed had very different m/z ratios in each mode, indicating the presence of co-eluting species. From the MS data it is possible to perform some investigation into the identification of the antioxidants that were present in the extracts. The m/z ratio of each of the peaks identified in the MS chromatograms was compared to the molecular masses of known antioxidants. If a match between the m/z ratio of the peak and the molecular mass of one or more antioxidants was found, this was considered a possible identification for that peak. For example, both lemon myrtle extracts show peaks with a m/z ratio of 139 Da indicating that the peak may be due to hydroxybenzoic acids.

However, positive identification is impossible without additional information such as MS/MS data and/or the comparison of the peaks with standard solutions. Due to the non-specific nature of the MS data that was collected in the study, only preliminary identification of the peaks could be performed. Thus a number of peaks could either not be identified or were identified as one of a number of possible antioxidants.In recent decades, advances in the final frost dates of winter or early spring have been observed throughout North America while advances in the timing of flowering have been documented in many angiosperm taxa . In response to recent climate warming, the flowering times of many species have changed, which may alter the risk of reproductive structures being exposed to spring frosts . Exposure of reproductive tissues to frost is hazardous for many plant species, as floral tissues are often the most vulnerable to frost damage, and the exposure of floral tissues to frost or freeze events can reduce pollen and seed production or result in reproductive failure . Over multiple generations, reductions in reproductive success due to increases in frost exposure may lead to progressive declines in local abundance, potentially resulting in local extirpation . Accordingly, the ability to initiate and to complete flowering and fruiting without exposure to frost or freeze events plays a major role in determining the geographic range of many species . Previous studies have predicted that progressive warming could increase the risk of frost damage to floral tissues for many species if,in response to warming, flowering times advance more rapidly than the date of last frost, defined as the date that marks the beginning of that portion of each year during which daily minimum temperatures remain above 0°C . This pattern has been particularly well documented among shrub and forb species whose flowering time is primarily driven by snowmelt , resulting in reductions to annual flower and seed production . Conversely, warming climates may advance the date of last frost more rapidly than plant species advance their flowering times, thereby reducing their risk of frost exposure ; this pattern has been detected among 14 European angiosperm species .

Warming conditions may also delay bud break and flowering of those taxa that require an extended period of winter chilling to break dormancy, protecting them from flowering prior to the onset of the frost-free period . While the phenological responses of flowering time to climate warming have been measured in thousands of species , and broad-scale temporal reductions in frost risk to developing leaves have been detected among North American trees , no large-scale examinations of shifts in frost risk have yet been conducted on a sufficient array of taxa to detect or to characterize general trends in a continental flora. As a result, the general effects of recent climate change on the risk of frost exposure to floral tissues remain largely unknown. Additionally, flowering phenology has previously been documented to be evolutionarily conserved among co-occurring taxa that are closely related . Given that exposure to frost depends on a species’ phenology at a given location, it is also possible that frost risk is phylogenetically conserved. However, no systematic examination of the degree to which frost risk is phylogenetically conserved among closely related taxa has yet been conducted. To address these gaps, we conducted the first continent-wide assessment of frost risk by evaluating the flowering times of 1,653 species collected in flower from 1920 to 2015 and represented by 475,694 digital records of herbarium specimens collected throughout North America, with specimens primarily concentrated in the Western and Eastern United States. By comparing rates of temporal changes in dates of last frost experienced by each species among the sites where it was sampled to rates of temporal changes in flowering date from 1920 to 2015, we determined that, for most species, the advancement of the last frost date has outpaced the advancement of flowering date, resulting in a reduction in the risk of floral exposure to frost. Furthermore, this pattern persisted across regions that historically experienced both early and late dates of last frost. We also conducted a phylogenetically informed analysis to determine whether,as has been found for flowering time itself , the risk of exposure to frost exhibits a phylogenetic signal. Finally, we compared the degree of frost risk experienced by native versus exotic species, and evaluated whether the relatively low risk exhibited by the latter is due to differences in the mean climate conditions they occupy or to differences between natives and exotics in the degree of phenological change that they exhibited.Phenological data pertaining to flowering times in this study consisted of 475,694 specimen records of angiosperm species collected in flower. These data were derived through filtering of a larger dataset consisting of 894,392 specimen records accessed from the digital archives of 72 herbaria , and cleaned using several criteria described below.

Estimates of mean flowering date from herbarium specimens have been reported to provide accurate estimates of species’ flowering times and have yielded estimates of phenological change similar to those derived from in situ observations of living plants across both temporal and spatial climate gradients . To ensure the quality of the data used in this study, large plastic pots for plants specimens were included in the dataset analyzed here only if, at the time of digitization, herbarium personnel had: verified that the specimens were collected when in flower; recorded GPS coordinates of the location from which the specimen was collected; and provided the precise date of collection . Only those specimens that were explicitly recorded as being in flower within either the DarwinCore “reproductivecondition” or “lifestage” fields of their source’s database were included in this study. Specimens that were listed only as “buds present” or “fruiting” were not considered to be in flower for purposes of this analysis, as some perennial species collected during the winter may be described as “buds present” when buds are completely dormant, or may retain aborted or unripe fruits that cannot be distinguished from recently matured fruits preserved on herbarium specimens. The taxonomic nomenclature used to identify all specimens, which sometimes changed over time or differed among collectors, was standardized according to The Plant List and TROPICOS using the Taxonomic Name Resolution Service iPlant Collaborative, Version 4.0 and subsequently filtered to eliminate all taxa not identified to species level within the megaphylogeny used by the PhyloMaker package in R , which similarly used a standardized taxonomy derived from TPL and TROPICOS . To avoid pseudoreplication, duplicate specimens were also removed. The resulting dataset included 475,694 specimens representing 1,653 species distributed throughout North America . In this study, we calculated the frost risk of each sampled species using annual estimates of the date of last frost at each collection site obtained from ClimateNA version 5.5.1. Frost risk of each species was defined as the proportion of its specimens collected in flower before the date of last frost in the years and locations in which they were collected. Frost risk in this context does not invariably predict the risk of reproductive damage, which depends not only on species- and population-specific cold tolerances, which are undocumented for most taxa , but also on microclimate conditions that cannot be easily incorporated into continental-scale datasets, such as humidity, wind speed, and recent precipitation . Nevertheless, temperatures of 0°C have been documented to damage floral tissues ofa wide variety of species , as radiative cooling often results in damage to floral tissues and emerging leaves under nighttime temperatures of 0°C even in species that otherwise remain hardy to subzero temperatures . Thus, frost risk is used here as a standardized metric indicating the likelihood of exposure of floral tissues to frost or freeze events.To estimate historical frost risk for each species, we calculated the proportion of specimens of each species collected from 1920 to 1979 that were collected prior to the date of last frost at the site and year of their collection. To estimate recent frost risk, we similarly calculated the proportion of specimens of each species collected from 1980 to 2015 that were collected prior to the date of last frost at the site and year of their collection . To ensure that a sufficient number of observations of each species were available to produce meaningful estimates of frost risk within both periods, we eliminated all species that were not represented by at least 50 specimens both prior to the year 1980 and after the year 1979.

A large number of these genes were identified as differentially expressed over the course of fruit development, which is consistent with previous studies of transcriptome changes during fruit ripening in sweet orange . However, most genes showed similar temporal expression patterns among all rootstock genotypes. Furthermore, only ~15% of the genes were genotype-specific . Therefore, the remainder of this study focused on DEGs identified between these rootstock genotypes during fruit development. A total 684, 388, 361, 178, 395, and 885 genes were significantly differentially expressed between RL vs SO, CZ vs SO, TF vs SO, RL vs CZ, TF vs CZ and TF vs RL rootstocks respectively . The majority of the differentially expressed genes are observed in comparisons involving rough lemon rootstocks, especially compared to trifoliate orange. This is consistent with the observed differences in fruit quality traits, as fruit of trees grafted on rough lemon rootstock showed consistent significant differences from fruit of trees grafted on the other three rootstocks in many of the traits measured . These results suggest that rough lemon and trifoliate rootstocks show the greatest effects on the scion and are good candidates to identify graft-related genes playing a role in fruit quality. The largest and most significant changes in gene expression between rootstocks were observed at time points two and three . Among the DEGs were several genes with functions involved in fruit quality traits, such as those relating to starch and sucrose metabolism, fructose metabolism, and hormone signaling related genes. KEGG pathway analysis displayed plant hormone signal transduction, carotenoid biosynthesis, plastic pots for planting and fructose and mannose metabolism pathways to be significantly enriched. Several genes involved in various hormone-signaling pathways were DE, mainly genes in the abscisic acid and auxin-response pathways.

Several genes involved in these pathways were chosen to validate the RNA-seq data by qRT-PCR due to their potential biological significance regarding rootstock effects on fruit quality.ABA has been known to be a regulator of fruit ripening and response to abiotic stress in non-climacteric fruit. AHG1, a homolog of Arabidopsis PP2C family protein, was DE in this study. PP2C is a negative regulator of the ABA hormone-signaling pathway. This gene was slightly up-regulated when comparing fruit of trees grafted on trifoliate to fruit of trees grafted on rough lemon rootstock at time two and significantly down-regulated at time three . Upregulation of AHG1 is in accordance with previous studies showing this gene being induced by water stress, which may have occurred in September. The downregulation of this gene later in the season could be correlated with increased fruit maturation in fruit grown on trifoliate rootstocks. This is in agreement with a study in tomato where suppression of PP2C expression led to increased ABA accumulation and higher levels of ABA-signaling genes that increase the expression of ABA-mediated ripening-related genes.Auxin signal transduction is mediated by Aux/IAA and ARF genes. Aux/IAA proteins are negative regulators of the auxin signal transduction pathway. In this study, a gene encoding an Aux/IAA protein, IAA16, was up-regulated in fruit grown on trifoliate compared to rough lemon rootstocks at time two and three . A previous study revealed that a gain-of-function mutation in IAA16 displayed reduced response to auxin and ABA, which led to reduced plant growth. Silencing of related Aux/IAA genes increased fruit size in tomato due to auxin control of cell expansion and elongation. In addition to Aux/IAA, another early auxin-response gene, SAUR78, was DE in this study. This gene was down-regulated in fruit grown on trees grafted onto trifoliate compared to rough lemon rootstocks at time two and three . Small Auxin Up RNA genes are a group of auxin-inducible proteins.

Other SAUR genes have also been shown to promote cell expansion. Furthermore, a MYB77 gene encoding a transcription factor was DE in this study, displaying a slight increase in expression in fruit grown on trifoliate rootstock at time two, but a large decrease in expression at time three . This gene was previously described as a regulator of the auxin signal transduction pathway. This protein was shown to interact with ARFs to promote plant growth. Interestingly, the effects of MYB77 in Arabidopsis were found to be increased by endogenous exposure to ABA and further promote plant growth. While these two studies were performed in roots, this transcription factor was shown to be involved in citrus fruit ripening, where it was highly correlated with ABA and suggested to have a similar function in response to the hormone.Although there were not statistically significantly differences seen in other genes in the auxin- and ABA-signaling pathways, trends could be observed during hierarchical clustering of these genes. Many of the genes within a family shared common expression levels and generally follow the predicted regulatory patterns in their respective pathways . Taken together, the changes in ABA- and auxinresponsive genes suggest a potential mechanism for induced ripening by trifoliate rootstock and larger fruit produced when rough lemon is used as a rootstock.The expansion phase of citrus fruit development involves cell enlargement and water accumulation. Given the changes in hormone-signaling pathways that likely lead to changes in fruit size, other genes related to fruit growth, such as transporters and genes related to cell wall metabolism were investigated. This led to the identification of two DEGs that could be influencing fruit size. The first, a Plasma membrane Intrinsic Protein 2 gene encoding an aquaporin was down-regulated in fruit grown on trifoliate rootstock . Water import in plants is mediated by aquaporins and essential for cell expansion.

These genes were highly expressed in expanding green grapes and one was identified as a candidate gene under the QTL for berry weight. PIP genes were also associated with an increase in volume of fruit in apple and strawberry. The second DEG, an expansin , was also down-regulated in fruit grown on trifoliate rootstock . Expansins play various roles in fruit development, including cell elongation and cell wall softening. A homolog of EXP1 in tomato was expressed during green fruit cell division and expansion with maximum accumulation of EXP1 during the late phase of green fruit expansion and early maturation. The increase in expression of these two genes in fruit grown on rough lemon rootstock could contribute to the larger fruit size observed. In addition to cell division and cell expansion, during fruit development, fruit softening is also an important feature that relies on cell wall metabolism. The Trichome Birefringence-Like gene, which encodes a protein required for cellulose biosynthesis, was identified in our study as DE. Mutations in this gene caused a reduction in the amount of pectins and an increase in pectin methylesterase activity. PME catalyses the demethylesterification of pectin, which may undergo depolymerisation by glycosidases. TBL23 was up-regulated in fruit grown on trifoliate rootstock compared to rough lemon , suggesting a potential role in fruit softening during citrus ripening. Transcription factors also play an important role in plant development and fruit ripening. Several transcription factors were differentially expressed in this study. GO enrichment showed the molecular function GO term ‘DNA-binding transcription factor activity’ was significantly enriched. In addition to the MYB77 transcription factor gene described earlier, a GRAS transcription factor gene, HAM3, was DE in this study. GRAS transcription factors were previously found to play a role in berry development and ripening in grapes, tomato, and citrus. This transcription factor showed increased expression later in the season when fruit were grown on trifoliate rootstock, drainage for plants in pots suggesting the rootstock influences its role in improved citrus fruit quality.The largest phenotypic differences seen in mature fruit grown on trifoliate compared to rough lemon rootstock were in the levels of total soluble sugar and titratable acid in ripe fruit. The levels of sugars and acids and their ratio in fleshy fruits is one of the most important determinants of sensory traits such as taste and flavor. Two genes were identified as differentially expressed that could play a role in the accumulation of these compounds. Firstly, a P-type ATPase was DE in fruit growing on trees grafted onto trifoliate versus rough lemon. This gene was down-regulated at time two, but upregulated at time three . Studies have proposed a number of ATPases as proton pumps that are responsible for organic acid accumulation in citrus fruit.

The reduced expression of this ATPase gene later in the season in fruit grown on rough lemon rootstocks could contribute to the lower accumulation of titratable acid levels in these fruits. This ATPase gene identified in this study was not identified in the previous citrus studies, but the regulation of acid accumulation is a complex, as can be seen in other fruits, such as papaya and apple. It is possible this is a graftinduced effect observed with these specific rootstocks, which were not examined in the previous studies. Secondly, a homolog of Arabidopsis BETAFRUCT4 was down-regulated in fruit of trees grown on trifoliate rootstock compared to rough lemon at time three . This gene encodes a vacuolar invertase. Decreased expression of vacuolar invertases has been associated with increased sucrose content and accelerated ripening. Interestingly, by using an antisense acid invertase gene in transgenictomato to reduce acid invertase activity, fruit displayed higher levels of sucrose, as well as smaller fruit. We see similar trends in sugar accumulation and alterations in fruit size in this study. Klann et al. suggested that the water influx that drives fruit expansion is closely related to the concentration of osmotically active soluble sugars and therefore, all genotypes accumulate water until they reach a similar threshold of soluble sugar concentration. This could also contribute to the increased size of fruit grown on rough lemon fruit compared to trifoliate rootstocks.This study did not identify any statistically significant differentially expressed miRNAs from our fruit small RNA seq data. Therefore, potential miRNAs that target DEGs were predicted. An in-house R-script was used to select for miRNA-mRNA interaction pairs with an expected negative correlation in gene expression. These pairs were identified for the ten genes described above. All ten miRNA genes and their target mRNAs were detected by qRT-PCR. Pearson correlation coefficient value between the relative expression level detected by qRT-PCR and by RNA-sequencing was highly significant with r = 0.94. Of the ten interaction pairs, eight followed expected fold changes between timepoints . Therefore, it is likely that these eight target mRNAs are being regulated to some extent by their respective miRNA.Citrus is now grown in more than 140 countries in tropical, subtropical and Mediterranean regions. It is one of the most economically important crops in the world. Citrus are rarely grown from seed and virtually all commercial citrus is propagated by grafting. This reduces the juvenile phase, allowing for the trees to produce fruit many years earlier than would trees grown from seed1 . Due to the large variation in growing conditions and climate in the regions where citrus is grown, different citrus rootstocks are required to improve yield and fruit quality in numerous diverse climates, as well as resist various pests and diseases. Rootstocks impart certain traits to the scion and the effects of rootstocks can be large. The most significant impacts are on growth, vigor and yield, tree nutrition, stress resistance, and fruit quality. The rootstock effects on various aspects of tree growth and fruit development are well documented, but the molecular mechanisms underlying most of these differences are unknown. Previous studies have shown changes in the transcriptome of various rootstock genotypes, especially in response to biotic and abiotic stressors. These types of changes have been seen in Arabidopsis, corn, mulberry, tomato, and poplar. In citrus, gene expression profiling has been used to understand rootstock effects and responses to biotic and abiotic factors. In another study, expression studies of leaves from mandarin grafted onto various rootstocks were analyzed in order to explain rootstock effects on the growth of scions. There is extremely limited tissue-specific transcriptome knowledge in citrus, especially for root tissue. A small number of studies have evaluated trifoliate, trifoliate hybrid, and mandarin root transcriptomes in response to citrus diseases, but these studies each assessed only one genotype. Only recently has an RNA-seq based approach been used to establish a reference transcriptome for citrus and of the 28 samples used in the study, only two were obtained from roots.

Urban ecosystems are temporally dynamic systems, yet historical factors associated with land use legacy and time lags as a result of development have largely been overlooked. Because human-altered landscapes are relatively recent, it is particularly important to recognized that the observed biodiversity may be undergoing a process of change from the previous land use type to the new one when interpreting observations. Just as urban ecosystems are dynamic across years, they are also dynamic intra-annually, with resulting phenological shifts within urban landscapes compared to surrounding natural habitat. The urban heat island effect is a well-documented phenomenon where the city environment can be significantly warmer than the surrounding landscape as a result of impervious surface area that retains heat and higher energy usage, causing changes in the timing of ecological patterns. As a result, plants bloom earlier the more densely urban the surrounding habitat is and bird migration advances earlier in urban contexts. Another potential effect of land use change in urban and agricultural landscapes beyond climatic can be the variety and timing of floral resource availability. Urban areas, while having less green space, often grow many exotic plants which are supplemented with water and nutrient inputs that allow for an extended flowering season. As a result, urban areas are characterized by relatively low , but constant, floral resources throughout the year. Agricultural landscapes have large patches of dense, often homogenous, floral resources that will fluctuate greatly from early spring to the end of the summer due to mass-flowering monoculture crops. In contrast, pot with drainage holes many natural areas in California experience a large burst of diverse floral blooms in the spring, and by the end of the summer, there are very few floral resources available.

Bees provide the majority of animal mediated pollination services on which an estimated 87.5% of flowering plants depend . The value of pollination in agriculture is estimated at $200 billion worldwide, largely due to many foods that are essential for food security and a healthy human diet, including numerous fruits, vegetables, and nuts that require bee pollination. In addition, there has been growing interest in urban agriculture to ensure food security and access to healthy foods for urban populations. One study estimated the economic value of urban fruit trees in the one city of San Jose, California to be worth $10 million annually. Honey bee populations and many bumble bee species are declining worldwide, while many other bee species have not been closely documented enough to determine their status. One of the reasons proposed to be negatively affecting bee populations is land use change. A review of 265 papers studying the effect of land use change on pollinator populations found more negative than positive impacts with a wide window of variability. This could partially be attributed to the wide diversity of pollinators themselves, as well as the varying definitions that people use to constitute land use change. We propose that an additional cause of high variability can be explained by investigating how the seasonal patterns of bee communities may shift in different neighboring land use types which experience highly different availability and suites of floral resources. Although bee seasonality has been documented in urban and agricultural landscapes, no studies to our knowledge have specifically investigated the differences in seasonal patterns of population abundances of bees between human-altered landscapes and neighboring natural habitat, despite the established seasonality of bees and variability of floral resource availability.

Bees are often sampled throughout the season, but all of these data are typically lumped together, potentially obscuring subtleties in change . Here, we investigate how local bee communities shift over the course of the flowering season in urban, agricultural, and natural land use types. We make use of a “natural experimental design” in which urban, agricultural, and natural areas intersect in a peri-urban landscape on the outskirts of the San Francisco Bay Area in Contra Costa County, California. To study the impact of changing land use on local bee community population dynamics, we sampled the bee community flying through the landscape at four time points over the course of the season for three years at 24 sampling locations.At each site we laid out a standardized pan trapping transect of fifteen 12 ounce bowls spaced 5 meters apart in alternating colors of fluorescent blue, white, and fluorescent yellow. Bowls were filled to the brim with soapy water . In 2010, transects were set up for a 4 hour period between 10:30am to 2:30pm , with 4 sites sampled per day, and all sites sampled on consecutive days. These 2010 transects were run twice, once in the early summer, and once in the late summer. In 2011 and 2012, sampling was conducted over a 24 hour period, so that more sites could be run simultaneously and more samples could be collected per site per year. All 24 sites were sampled within two consecutive collecting windows , and were run four times each year: early spring, late spring, early summer, and late summer. Because we were interested in landscape level effects, we tried to control local variables as much as possible. All sites were selected in easily accessible, open areas that received full sun. Natural areas were in grassland habitat, so we selected agricultural sites that were either weedy field margin edges or fallow fields, and urban sites that were vacant lots or green ways.

The human-altered sites were deliberately selected to not be adjacent to any mass flowering plants of agricultural crops or gardens. The goal of collection was to sample the bee community that was flying through the site searching for resources. Bees were collected from the pan traps by using a metal strainer, rinsed with water, frozen overnight or longer, and then pinned and labeled. Specimens were sorted to the genus level, and then to the species level with the assistance of Dr. Robbin Thorp , Professor Emeritus, UC Davis. The only exception to identification at the species level were bees of the genus Lasioglossum, large pot with drainage due to their overwhelming abundance, limited availability of taxonomic expertise for this group, and lack of known ecological diversity. Voucher specimens and the majority of the total collection will be deposited at the Essig Museum of Entomology at UC Berkeley.For response variables including aggregate bee abundance, species richness, Shannon diversity, and number of rare species, we tested for the effect of land use type, seasonality, and their interaction with generalized linear mixed models using the R package lme4. We designated collecting method , land use type, seasonality, and the interaction of land use type and seasonality as fixed effects, and site and year as random effects. We analyzed the effect of time both categorically by collecting period and continuously by day of year. Day of year was normalized on a scale of 0 to 1 from the first collecting date to the last. Shannon diversity was fit with a Gaussian distribution while all other variables were fit with Poisson distributions.We found that the bee communities in human-altered landscapes experienced different phenological patterns than the neighboring natural areas. Increased temperature in urbanized areas as a result of the urban heat island effect is often cited as the driving force for changed ecological dynamics. We propose another driver of local phenological shifts: the timing and quality of floral resource availability, due to irrigation that extends the flowering season throughwater inputs and landscaping choices in urban residential, public, and commercial zones, as well as mass flowering crops in agricultural fields. For example, Eucera actuosa was collected most frequently in human altered sites in the early spring and very little in late spring, whereas in natural sites it was collected in lower numbers in early spring and peaked in late spring. Another bee, Melissodes lupina , is a later flying bee than E.actuosa and experienced the opposite pattern. Melissodes lupina was collected most frequently in the early summer for natural areas, but was collected more often in the late summer in human-altered landscapes. Both E.actuosa and M. lupina demonstrate a pattern of relative abundance of species being shifted in different land use types. Even further, the temporal direction of relative abundance in both examples areas skew towards higher abundance during the middle of the season for natural areas and higher abundance at the more extreme ends of the season in the human-altered landscapes.

In other words, these are not simple patterns where species in human-altered landscapes always are collected earlier, which is generally the result of the urban heat island effect. Instead, this is likely due to irrigation effects extending flowering times in human-altered landscapes that provide bees with necessary resources for extended flight periods. This supports our theory that patterns of change in bee community distribution throughout the year are a result of the different land use types offering variable seasonal floral resources. While ecologists have used time as an important variable in many different systems, only recently has time begun to be incorporated into urban ecology. These differences in relative bee abundance throughout the year could be the result of resource tracking or a shift in emergence timing between different land use types. Shifts in plant phenology have been well documented in temperate urban landscapes. Urban areas have been associated with earlier plant blooming closer to the city center, and the urban heat island effect also can directly affect animal populations. In addition, historical temperature and museum collection records show a link between climate change and advancing bee emergences. Bees could be responding to local climatic differences or floral availability respectively, with both emergence timing and length of the flight season of bee species being impacted differentially at a micro-scale between different land use types. Pollinator responses to land use change are generally more often negative than positive, although there is high variability of outcomes due to many different experimental design types, systems, and the use of simple community metrics rather than more species specific analyses. Using functional groups such as nesting type, generalized foraging , and sociality, more patterns have emerged about traits that may be most sensitive to anthropogenic disturbance, although the type of disturbance will affect bees differently. For example, ground nesting species may be more successful in intensified agricultural landscapes, while cavity nesting species may be more common in urban landscapes because of increased nesting resources. We had 7 species that were collected almost exclusively in human-altered sites . These species positively associated with anthropogenic change covered a range of functional groups for sociality, nesting type, foraging generalism, size, and distributional range. Two of these are non-native , and as a group they have a wide diversity of life history traits. For example, Ceratina dallatorreana, a species of small carpenter bee originally from theMediterranean region first collected in California in 1949, has the unusual life history trait of female parthenogenesis in its local population here. Megachile rotundata is another nonnative bee accidentally introduced to the United States from the Mediterranean , is a solitary leaf cutter species that have become an important managed pollinator in the Western United States. Andrena chlorogaster, is a native California bee, with a generalized life habitat and wide range, while the squash bee, Peponapis pruinosa, is a solitary specialist on cucurbits. These species that favor human-altered landscapes not only fail to share many life history traits, but they are also diverse phylogenetically, comprising several different bee families. These different patterns of bee distributions could be the result of two possibilities: either bee populations are tracking resources between the different land use types, or the bee communities in the different land use types are experiencing different emergence timing. Bee movement has been notoriously difficult to study because of the size, mobility, and life history of this group. Some species and system-specific conclusions about bee foraging distances and size based models of foraging distances have been made. However, these are all based on foraging movements anchored around a central nest that a female bee is provisioning, and in contrast almost nothing is known about dispersal movement—in other words, how far bees might travel from their emergence site to mate and select their own nest site.