Category: hydroponic agriculture

Model plants used to better understand plant defense gene response to P. cinnamomi infection include; Zea mays, Arabidopsis thaliana, Lupinus angustifolius,Castanea sativa , Eucalyptus nitens, Lomandra longifolia, and most recently N. benthamiana . The gene expression in susceptible model hosts such as L. angustifolius and N. benthamiana can be compared to tolerant hosts like A. thaliana and L. longifolia to identify differences that may be associated with resistance to P. cinnamomi. Santos et al compared the gene expression between a susceptible and resistant variety of chestnut. They found that genes encoding for proteins involved in pathogen recognition proteins , were significantly upregulated in the resistant variety especially before inoculation. Six out of eight defense related genes including; WRKY31 and LRR-RLK’s were more highly expressed in the uninoculated C. crenata when compared to the uninoculated C. sativa. This increased basal defense to P. cinnamomi may contribute to this variety’s resistance. Gene expression in E. nitens in response to P. cinnamomi infection included up-regulated over represented gene ontology terms related to JA and ET signaling . Interestingly, pathogenesis-related gene 9 was down-regulated and represents a cross-species effector target during P. cinnamomi infection. Functional genomics and validation of these defense genes has only been performed in one study in A. thaliana. Eshraghi et al. reported that an auxin Arabidopsis mutant was more susceptible to P. cinnamomi infection than the wild type indicating the role of auxin pathways in P. cinnamomi defenses.The main challenges for the identification of P. cinnamomi resistance genes in avocado are the lack of tools available for functional genomic studies and limitations associated with tree crop biology.

Next-generation sequencing has provided some information on the expression of defense-related genes in avocado infected with P. cinnamomi. However, 25 liter plant pot the lack of the genome sequence and absence of functional genomic tools for avocado makes it difficult to determine and confirm their contributions to resistance against P. cinnamomi. The N. benthamiana model plant provides the opportunity to conduct functional genomic studies to determine the role of defense response genes to P. cinnamomi resistance that is not yet available in the avocado system or other tree hosts. Model plants including A. thaliana , L. angustifolius , and Medicago truncatula have been previously reported as susceptible hosts for this oomycete pathogen and have been used to study P. cinnamomi pathogenesis and plant responses to this pathogen. Although whole genome sequencing was available for these pathosystems, functional assays were not conducted with the exception of one study in Arabidopsis implicating the auxin signaling pathway with defense response against P. cinnamomi . Conducting RNAseq studies in N. benthamiana system at different time points during the infection process will provide a foot-hold into the defense gene expression pattern during P. cinnamomi infection and will allow us to conduct functional studies of selected defense genes using this N. benthamiana–P. cinnamomi pathosystem. Differentially expressed pathways and genes can be then validated by RT-qPCR in N.benthamiana and in avocado inoculated with P. cinnamomi using a detached leaf assay. Functional validation of the most promising genes can be done in N. benthamiana by transient over expression or silencing to determine their contribution to P. cinnamomi resistance. If similar expression patterns are found in avocado it is reasonable to consider this gene a good candidate for marker assisted breeding or biotechnology in avocado.

As genomic tools for avocado quickly become more available the methods developed in this system will become more applicable to this fruit tree crop. RNAseq analysis of infected N. benthamiana roots can complement this system by identifying what genes are universally expressed in the plant in response to P. cinnamomi infection and what gene expression is unique to the roots. Functional genomics are lacking in avocado; therefore, the objectives of this study were i) to establish a model system to look at defense gene expression in response to P. cinnamomi infection, ii) validate differentially expressed defense genes using overexpression in the same N. benthamiana model system, and iii) establishing connections to similarly expressed defense genes in avocado in response to P. cinnamomi infection. This information will help to select candidate defense genes in avocado for marker assisted breeding or biotechnology.Carbohydrates, mainly sugars and starch, are the major reserve, flavor, and textural components of many horticultural crops, and as such, determine their nutritional value, postharvest quality, and storage life. However, even in seemingly ‘carbohydrate-irrelevant’ leafy greens and various produce, starch and sugars may exert less obvious, yet we argue, critical roles in shaping post harvest quality. Starch and sugars have diverse functions in cells depending on their relative concentration, mobilization, subcellular location, and interaction with proteins. They sustain growth and buffer cells from stress, and as signaling molecules, they regulate many pathways that determine nutrient allocation to the sinks, and their partitioning into different biomolecular pools. Here, we intend to build several theoretical frameworks to show that carbohydrates, especially starch, may have ‘surprising’ roles in determining horticultural postharvest quality. We show that starch may be essential to, and intertwined with, climacteric ripening of fruits; starch is a determinant of leafy-green shelf life; sugars can influence the synthesis of specialized ‘sensory’ compounds; carbohydrates have roles in biotic and abiotic stress response and in determining fruit size; and that source tissue can determine sink quality.

We then point to molecular targets that can alter the carbohydrate profile of produce to obtain desirable traits.Starch and sugars accumulate in many fruits, tubers, and leaves, but with different temporal patterns and consequences for postharvest shelf life and quality . The timeframe over which the flux between starch and sugars occurs determines the classification and the role of carbohydrates. Harvested produce is often stored in the dark and at low temperatures, where respiration of reserves sustains the hexose phosphate pool . When reserves are exhausted, carbon starvation triggers senescence that manifests as spoilage. In tissues that accumulate starch as a carbon and energy reserve , granule degradation to sugar is surprisingly complex and multilayered. First, starch degradation occurs simultaneously with its biosynthesis at the granule surface even during the phase of net starch accumulation. This permits a bidirectional flow of carbon from starch to sugars, and potentially, to other compounds viathe hexose phosphate pool . Second, in fruit, in addition to exocorrosion at the surface, there is evidence of some endocorrosion in the starch granule, during ripening and fruit cold storage. Understanding the bidirectionality of flux between starch degradation and synthesis, and the physical organization of the starch granule associated enzymes, may offer opportunities to adjust reserve utilization during postharvest storage .The limited carbohydrate levels in leafy- and microgreens influence postharvest longevity by buffering against senescence. Shelf life is extended when harvested leaf starch levels are high, such as at the end of the day or after an extended light period. Starch content also positively correlates with desirable attributes such as sugar content, fresh weight, and texture. Identifying mechanisms that potentially coordinate photosynthesis, carbohydrate content, respiration, texture, and postharvest longevity in leafy greens is needed.Among fruits, the accumulation of high levels of starch appears to be a unique feature of those with climacteric ripening. In tomato fruit, starch may provide ∼40% of the carbon needed for fruit respiration. Furthermore, some of the fruit’s internal CO2 from respiration is likely fixed by fruit chloroplasts, contributing to ∼10–15% of ripe fruit carbohydrates. Transitory-storage starch may represent an evolutionary strategy for reproductive fitness with unintended benefits for the postharvest industry. First, its biosynthesis in climacteric species likely amplifies fruit sink strength, to undergird sink establishment and productivity. A large difference in sucrose concentration between source and sink, black plastic plant pots which occurs when imported sugars are converted to starch, would enable higher carbon allocation to fruit. Further, carbon storage as starch rather than as sugars minimizes cells’ osmotic disturbance. Second, increased fruit starch biosynthesis may also enhance plant survival under stress . From a postharvest perspective, the starch in climacteric-ripening fruit may be a vital energy source for maintaining biological processes, and for the synthesis of ‘quality-related’ metabolites that would minimize loss and waste . In contrast to the fruit described above, ‘sugar-storers’ are mainly non-climacteric and accumulate comparatively little starch . Furthermore, the starch is deposited in the peripheral regions of the fruit, and unlike the climacteric fruit, its accumulation peaks and is degraded to sugars, early in fruit development. However, starch in ‘sugar-storers’ may still contribute to fruit growth and quality by enabling a higher import of sugars into the developing fruit.Starch content and composition directly determine the functionality of starch in staple roots and tubers, but presumably, can also influence the biological processes of fruits and vegetables, which do not accumulate high levels of starch, as shown below: Produce firmness The crystalline and insoluble nature of starch directly contributes to the firmness of fruit and the texture of leafy vegetables.

When the dense granule is degraded to soluble sugars, intercellular space increases, thus promoting tissue softening. Tuber nutritional quality and textural attributes The relative proportion of the amylose and amylopectin fraction of starch is critical to tuber nutritional status and textural properties. Amylose is resistant to digestion and simulates fiber in the intestinal tract. Since amylose improves the nutritional value of starch-rich commodities, there have been many biotechnological efforts to increase its proportion relative to amylopectin in crops. Furthermore, the molecular structure of amylose is such that if high-amylose potatoes, cassava, and so on, are fried, they should have a crisper texture that may be desirable to consumers. In contrast, amylopectin provides smooth and moist textures to cooked starches which may be suitable for other end-uses. Sugar availability in fruit Starch granule crystallinity, composition, morphology, and size collectively influence starch degradation to sugars, which in developing fruit could have consequences for fruit respiration, metabolism, and ripening. We propose that the digestion of starch to sugars may be an inflection point for the rate of reserve use during ripening. Engineering starch with an optimal composition and crystalline structure to control the rate of release of sugars, may be valuable in regulating fruit metabolism, and hence quality.The relative sweetness of sugars varies as such: fructose > sucrose > glucose > sorbitol, so if the sugar content is the same, different proportions of sugars will give the fruit distinct taste and flavor profiles which are prime concerns for consumers. Modulating fructokinase activity and the SlSWEET sugar transporters in tomato increased fructose content, silencing A6PR reduced sorbitol relative to glucose in apple, and modifying PuWRKY31, promoted sucrose accumulation in pear. These changes should increase fruit sweetness, and, along with organic acids and aroma volatile compounds, should favorably influence fruit taste, flavor, and consumer likability.Sucrose and hexose have differential effects on fruit size, a trait determining consumer acceptance, shelf life, and nutritional characteristics. A high hexose-to-sucrose ratio in early fruit development stimulates mitotic activity that increases cell number through hexokinase signaling. Additionally, the higher osmotic potential of hexoses relative to sucrose will attract more water, increasing cell volume. Genes influencing fruit size, mediated in part by changes in carbohydrates, include the SWEET phloem sugar transporters in tomato and cucumber, the CsSUS4 gene in cucumber , and the SlCDF4 transcription factor and its Arabidopsis homolog in tomato. In apples, the sugar-to-acid ratio correlates with fruit size, which may have been selected through domestication. Although the mechanisms underlying these phenomena may vary, they show that carbohydrates are key determinants of fruit/organ size.Sugars both fuel and regulate the accumulation of specialized metabolites that are important to postharvest quality. Switches in the flux of carbon between primary and specialized metabolism were seen when sucrose was added exogenously to strawberry fruit, which inhibited the expression of many carbohydrate genes, but stimulated the MYB5 TF that regulates anthocyanin levels. Changes in flux were also seen when ectopic expression of the AtMYB12 TF in tomato reprogrammed carbon away from primary metabolism and toward flavonoid biosynthesis via the shikimate and phenylalanine pathways. There is a clear interrelation between primary and specialized metabolic pathways, but it remains relatively under investigated in horticultural crops. Identifying and modulating the TFs that regulate the fluxes between these pathways would enable the design of plants with a desirable combination of primary and secondary metabolites.Changes in plant carbohydrates, including the starch-tosugar conversion, are an important acclimatory response to stress, often with consequences for produce quality. For example, sugars maintain cells’ osmotic potential, provide energy for stress defense, and act as membrane protectants and ROS scavengers, as shown below.

We calculated each effect size in R using the escalc function in the ‘metafor’ package . We fit meta-analytic and meta-regression multilevel linear mixed-effects models, using the rma.mv function in the ‘metafor’ package . We used three random effects to control for non-independence of effect sizes collected from the same study or plant species: study ID, plant species, and an observation-level ID for individual SVE measures. We used phylogenetic comparative methods to account for non-independence that may arise due to shared evolutionary history of focal plants by including a phylogenetic covariance matrix. The phylogeny and branch lengths used to compute a phylogenetic covariance matrix came from a recently published, dated megaphylogeny contained in the package ‘V.Phylomaker’ , which combines the seed plant phylogeny from Smith and Brown with the pteridophyte phylogeny from Zanne et al. . Despite slightly higher AIC values and larger P values , we present results from models including phylogenetic controls to fully account for non‐ independence due to shared ancestry . Both analyses produced qualitatively similar results and neither the magnitude nor the sign of SMD estimates changed when phylogenetic controls were included . However, uncertainty around SMD estimates was consistently smaller in models without phylogenetic controls, such that marginally significant effects became significant when phylogenetic controls were removed. Thus, inclusion of phylogenetic controls renders our analysis more conservative. With this mixed-effects structure, we specified four models, growing strawberries vertically which include an intercept only model , and three meta-regression models for different fixed effects/moderators: pollinator taxonomic group, whether the plant was a crop plant , and for native plants, whether it was in the honeybee’s native range .

We follow Hung et al. and define the West Palearctic as the honeybee’s native range . For the analysis comparing honeybee comparative effectiveness inside and outside of the honeybee’s native range, we excluded non-native plants from the analysis.To test whether there was a relationship between a pollinator taxon’s single visit effectiveness and visit frequency, we calculated Pearson’s correlation coefficients for the relationship between visit frequency and pollinator effectiveness for each unique study, plant, site, and year combination in which there were at least five pollinator taxa represented. We filtered data because sample variances cannot be confidently estimated when fewer than five observations are used to calculate correlation coefficients. In total, 26 studies of 50 plant species had visit frequency and effectiveness data for at least five taxa, and 62% of studies were fully excluded. After calculating correlation coefficients, we used the escalc function in the metafor package to calculate Fisher’s r-to-Z transformed correlation coefficients and corresponding sampling variances. Using the same multilevel linear mixed-effects model structure and phylogenetic controls as described above we generated three models. The first model was an intercept-only model to test for the overall relationship between a pollinator’s visit frequency and single visit effectiveness. The second model compared three categories against one another: studies where honeybees were present, studies where honeybees were absent, and studies where we artificially removed all points corresponding to honeybees . We generated this third category to determine whether the patterns we observed were solely driven by honeybees themselves or whether there might also be indirect effects of honeybee presence on the relationship between visit frequency and single visit effectiveness. The third model tested whether there was an interaction between crop status and honeybee presence.

Our meta-analysis supports the hypothesis that honeybees are frequently not the most effective pollinator of plants globally. Across six continents and hundreds of plant species, honeybees showed significantly lower single visit effectiveness than the most effective pollinator . This general pattern is likely driven by comparison of honeybees against birds and other bees. The most effective bird and bee pollinators were significantly more effective than honeybees, as were the average bird and bee pollinators. The finding that birds are more effective than honeybees is based on only six studies that were likely focused on flowers frequently pollinated by birds. Nevertheless, it supports the idea that plants adapted to bird pollination have traits that enhance pollination by birds at the expense of pollination by bees . Although data for non-bee taxa were relatively sparse, honeybees were as effective as the average and most effective ant, beetle, butterfly, fly, moth, and wasp pollinators, confirming that non-bee insects can be important pollinators . Our results bolster initial work summarizing honeybee pollination effectiveness and demonstrate that honeybees are less effective than many other visitors and at best average. Analysis of crop plants also revealed important differences between honeybees and nonApis pollinators. Despite their abundance in commercial cropping systems, honeybees are less effective crop pollinators than the most effective bee pollinators and the average non-honeybee bees . This finding supports the idea that the importance of honeybees as crop pollinators derives largely from their numerical dominance as crop visitors .Our analysis adds robust evidence to a growing consensus that wild bees have the potential to contribute greatly to agricultural pollination. Indeed, wild bee species richness, functional diversity, and visit rates increase crop yield , and the use of managed honeybee hives might not compensate for losses in wild bee species richness and abundance .

For example, increases in honeybee visitation only occasionally increase crop pollination whereas wild insect visitation universally increases fruit set . As such, managed honeybees alone may be insufficient to meet the increased pollination demands of global agricultural production and our results validate the importance of actions to promote resilient native bee communities within agricultural lands . Honeybees were equally effective as pollinators of plants inside and outside of their native range and were less effective compared to the most effective other bees in both regions . This result is not entirely surprising based on what we know about the co-evolution of plants and pollinators. The non-honeybee bee community may contain specialists sympatric with their host plants. Meanwhile, if honeybees are broad generalists, selective pressure might be less consistent, even within the native range of honeybees. Furthermore, if the morphological features relevant to pollination are relatively consistent across plants within the same genus or family, insects may be capable of pollinating novel plant species. For example, Prunus spp. occur in Europe and North America and Osmia spp. are highly effective pollinators of Prunus tree crops in both regions , despite the fact that North American Osmia spp. do not have shared evolutionary history with the Prunus species introduced as tree crops.We found an overall positive relationship between visit frequency and single visit pollinator effectiveness, but this relationship was largely driven by data from systems in which honeybees were absent . The overall positive correlation suggests that more frequent visitors are also more effective, but this result should not be interpreted to indicate that visitation frequency is an adequate proxy for overall pollination importance . This positive correlation may suggest that pollinators which visit frequently do so to the exclusion of other plant species, such that they display high floral constancy. High floral constancy may indicate that visitors gather and transport more conspecific pollen . Although the pollen loads of visitors do not always adequately predict effective pollination , high conspecific pollen transport likely predisposes visitors to higher pollination effectiveness on average. Another possible explanation is that, drainage planter pot for pollen-collecting visitors, more frequent visitors could be more efficient at extracting large quantities of pollen and might therefore transfer more pollen depending on how well pollen is groomed. Addressing whether more frequent visitors transport more conspecific pollen or deliver fewer heterospecific pollen grains are ripe questions for further study. The finding that honeybees erode this otherwise positive correlation suggests that this hyper-generalist species is often a numerically dominant visitor with modest effectiveness and may modify the pollination context for plant communities. Interestingly, when comparing systems with and without honeybees, visit frequency and pollination effectiveness do not positively correlate even when we artificially remove the data on honeybees and re-calculate correlation coefficients. This result suggests that honeybee presence may indirectly influence the relationship between visitation frequency and pollination effectiveness by altering the visitation patterns and effectiveness of other plant visitors. High honeybee visitation frequencies may indicate that honeybees efficiently extract nectar and pollen without also efficiently depositing the pollen they extract . If honeybees deplete floral nectar, this could make plants less attractive to other common visitors and alter their visit behavior and effectiveness . If they extract large amounts of pollen , this could reduce the amount available for collection and deposition by other pollinators . Indeed, honeybees can outcompete and reduce visits from other pollinators, reducing wild pollinator abundance and the diversity of plant species visited by non-Apis species .

Honeybee competition can also decrease interaction diversity by causing pollinators to become more specialized . Such changes in plant-pollinator interaction patterns can ultimately reduce the reproductive success of plants species frequently visited by honeybees and change the pollination context for other species. There are several potential limitations of our study and possibilities for future work. First, we only included measures of female reproductive success in assessing pollination effectiveness . The proportion of extracted pollen that is successfully transferred to stigmas may be a better assessment of the overall reproductive contribution of different taxa , because pollen that is removed but not successfully transferred represents a loss to male fitness . Unfortunately, data on such transfer dynamics are much rarer in the literature. Second, there are likely other factors about plant and pollinator taxa that moderate the effects we observe but which we do not test in this study, for example, functional traits such as plant and pollinator specialism. We hope our study will motivate other researchers to pair our data with trait databases and information on single visit pollen removal to further investigate the factors that influence effective pollination.Managing urban runoff and its associated pollutants is one of the most challenging environmental issues facing urban landscape management. The conversion of naturally pervious land surfaces to buildings, roads, parking lots, and other impervious surfaces results in a rapid surface runoff response for both time of concentration and peak flow. Impervious land surfaces adversely impact the quantity and quality of surface runoff because of their effects on surface water retention, infiltration, and contaminant fate and transport. Large volumes of storm runoff from urbanized areas cause flooding, sewer system overflows, water pollution, groundwater recharge deficits, habitat destruction, beach closures, toxicity to aquatic organisms, and groundwater contamination. Traditional urban runoff management focuses on removing the surface runoff from urban areas as soon as possible to protect public safety. However, as excess surface water is quickly drained from urban areas, it is no longer available for recharging groundwater, irrigating urban landscapes, sustaining wildlife habitat and other uses. Green infrastructure uses natural or engineered systems that mimic natural processes to control stormwater runoff. For example, traditional detention ponds have been widely used to treat storm runoff and permeable paving promotes infiltration of rain where it falls. Importantly, decentralized green infrastructure strategies control runoff and contaminants at their source. Vegetation is a green infrastructure strategy that can play an important role in surface runoff management. Large-scale tree planting programs have been established in many cities to mitigate the urban heatisland effect, improve urban air quality, and reduce and treat urban runoff . There are municipal stormwater credit programs in a growing number of cities that promote retaining existing tree canopy, as well as planting new trees. Although these programs encourage planning and management of urban forests to reduce runoff impacts, fertilizer is required to promote plant growth, and these added nutrients may contribute to contamination of surface runoff . Thus, reducing nutrients in storm runoff is a challenging task for landscape and water managers. Bioswales are shallow drainage courses that are filled with vegetation, compost, and/or riprap. As a part of the surface runoff flow path, they are designed to maximize the time water spends in the swale, which aids in the trapping and breakdown of certain pollutants. Bioswales have been widely recognized as an effective decentralized stormwater BMP to control urban runoff. Their effects are threefold; vegetation intercepts rainfall reducing net precipitation; plant uptake of water via transpiration reduces soil moisture, thereby increasing subsurface water storage capacity, and root channels improve infiltration. New bioswales are being developed for harvesting surface runoff and supporting urban tree growth. Bioswales that integrate engineered soil mixes and vegetation are being used to enhance treatment and storage of surface runoff.

This result matches incredibly well with the predictions outlined in Figure 3.2. It also demonstrates that any spatial externalities associated with increased corn cultivation due to ethanol refinery location occur entirely within 30 miles of ethanol refineries. This suggests highly localized effects. What do the acreage increases highlighted in Figure 3.11 mean for nitrogen application? A 2007 Iowa State University Extension publication suggests that optimal nitrogen application for corn-after-soy is 125 lb N/acre, and optimal application for corn-after-corn is 175 lb N/acre . Taking a middle value of 140 lb N/acre, , the 298,718 acres of increased corn acreage estimated in Figure 3.11 represent 41,820,520 lbs, or almost 21,000 tons of extra nitrogen. These 21,000 tons of additional nitrogen that are attributable to the distance effect of ethanol refinery placement are essentially all applied to areas within 30 miles of an ethanol refinery. While this number is relatively small relative to the total application of nitrogen in the US Corn Belt, there is cause for concern about localized geographic effects. Nitrate runoff into local water sources is harmful to water ecosystems, animals, and humans, and has been a growing problem in the US Corn Belt . Local water quality data from the USGS could be used in future research to look for an effect of ethanol refineries on nitrate levels directly.The digitization of herbarium specimens and their associated data have advanced our ability to understand complex and changing biological systems . Digitizing herbarium records has advanced our ability to track changes in the distributions of organisms , stacking flower pot tower but herbarium specimens are rich with additional information regarding plant health, reproductive condition, and morphology that is generally not captured in current digitization workflows .

Because the utility of specimens for research is accelerating, it is essential that we structure digital data collection in ways that best facilitate longevity and integration across data sources. Of particular interest is the enormous potential of herbarium specimens as a resource for information on plant phenology . Plant phenology has complex, cascading effects on multiple levels of biological organization from individuals to ecosystems . Temporal mismatches between plants and pollinators can quickly drive populations extinct, cause rapid evolutionary shifts, and result in billions of dollars of agricultural losses . Phenology has also been used to study the impact of climate change in a range of organisms and vegetation types . Consequently, maximizing the use of herbarium specimens for phenological research is not only important for improving our understanding of evolutionary change, it is also a matter of great practical concern for addressing environmental problems. Recent studies have demonstrated the potential of herbarium specimens to be used in evaluating temporal and spatial variation in plant phenology despite known biases of herbarium records . These studies have provided three valuable outcomes. First, for several species, we now have a quantitative historical understanding of their phenological change over time . Second, for some species, relationships between temporal or spatial variation in phenology and climate have been detected; these relationships, in turn, provide a basis for forecasting the effects of ongoing climate change on the seasonal cycles of these taxa . Third, we have an improved understanding of the specific advantages of herbarium specimens for phenological research, such as filling gaps in long-term or observational data sets, either for a period of time or for underrepresented regions .

Given the ecological importance of phenology, the demonstrated value of herbarium specimens for phenological research, and the potential for digitization efforts to maximize herbarium records as a resource, it is necessary to develop robust standards for how phenological data are captured during or after the digitization process. There are currently two principal limitations to accessing and using phenological data from herbarium specimens: the paucity of high-quality images accompanying digitized specimen records and the lack of standardized methodology for capturing specimens’ reproductive traits and sharing the resulting data. If any phenological information is present on a label or visible on a specimen, it is parsed in numerous—and often arbitrary—ways during digitization. For example, phenological data embedded in a label might be “on a south facing slope in full flower,” but this information might be digitally captured in the ‘habitat,’ ‘notes,’ ‘plant description,’ or other field of a local database. Even if a local database does contain a field explicitly for phenological characters, each institution independently decides how to record the states present on the sheet. For example, in the SEINet collaborative , which consists of 251 U.S. collections and 11.8 million records, there are 2.6 million records with text present in a database field called ‘reproductive Condition.’ The majority of terms found within this field specify flowering, fruiting, sterile, spores, and/or cones; however, these terms are expressed in over 4000 unique text strings . Some collections specify “flowering,” while other records state “flws.” Some are ambiguous . The lack of a controlled vocabulary for this field makes aggregating these data for research purposes onerous.

Local databases often share their data with data aggregators such as iDigBio or directly with users as a suite of Darwin Core Archive files, an exchange standard described more fully below . However, the relevant Darwin Core fields are equally diverse, with most phenological traits being placed into the fields ‘occurrence Remarks,’ ‘organism Remarks,’ ‘dynamic Properties,’ ‘reproductive Condition,’ or ‘field Notes.’ It is clear that there is a huge potential for using phenological data from herbarium specimens . We propose a method here to broaden the scope and longevity of digitization efforts through a standardized methodology for scoring reproductive characters from herbarium specimens and provide a means of sharing the resulting data in a Darwin Core format. The protocol we describe here will unlock the potential of herbaria for phenological research by facilitating comparability among herbaria, research groups, and other methodologies used to collect phenological data .Prior to the workshop, we developed a survey to assess needs of the phenological community and herbarium data users and to review the current ways phenological data were being captured. We received 76 responses to the survey, and the respondents identified themselves as being from collections, monitoring, or research areas . With this survey and input from participants at the workshop, we reviewed the ways in which herbaria currently capture phenological traits. The two most-scored traits from specimens are the presence of open flowers and the presence of fruit . Most respondents also felt that of all possible traits, open flowers and fruits were the most important traits to score on a specimen. Participants of the workshop echoed this sentiment. We reviewed previous phenological research that was based on data derived from herbarium specimens in order to identify the types of raw data necessary and sufficient to achieve a variety of research goals. These findings are summarized in Willis et al. . When developing a scoring protocol, we considered the challenges and limitations to scoring specimens and the potential solutions to these limitations. We considered hard-to-see floral parts, trained vs. untrained scorers, the limited resources of most herbaria, and the likelihood of community-wide adoption. We also considered the costs and benefits of recording qualitative data vs. quantitative data . One of our primary concerns is that any resulting data from attempts to score phenological traits should be shareable in Darwin Core–formatted files to help ensure the usefulness and longevity of these data. Representatives from the data standards community, Biodiversity Information Standards , including Darwin Core and Apple Core, provided input for representing phenological stages using current biodiversity standards. Finally, to ensure that phenological traits from specimens can be integrated with other sources, participants included members of the USA National Phenology Network, the California Phenology Network, the National Ecological Observatory Network, the Royal Botanic Garden Edinburgh, the Pan-European Phenology Network, and the Plant Phenology Ontology. We propose that reproductive traits for specimens of seed plants be scored according to the following hierarchical categories/questions . Our protocol uses terminology from the Plant Ontology to represent plant parts  and traits that correspond to plant phenological traits in the Plant Phenology Ontology. By using a vocabulary that directly maps to ontologies, danish trolley data collected with this method can be easily ingested into data stores using those ontologies and thereby integrated with other sources of phenological data such as direct observations in situ or remote sensing .

The question “Are ‘reproductive structures’ present? ,” while the broadest question, was still determined to have value for scoring specimen records. Having this information allows researchers to filter millions of records quickly to find those that contribute to phenological research. It is also relatively easy for users with different levels of botanical training to score. A “yes” means that some reproductive structures of some kind are present. A “no” means that the specimen is sterile and strictly vegetative. It is important to note that this first-order scoring can apply to all taxonomic groups, even beyond seed plants. Some taxonomic groups may exhibit specialized structures that make it more difficult for non-experts to complete this process , but we anticipate that this challenge will be limited. Minimally, first-order scoring will allow for records to be filtered and then subsequently scored in more detail.For specimens that are scored as having reproductive structures present, it is valuable to characterize which reproductive structures are present. Most research thus far has used specimens with open flowers. For flowering plants, we propose the following second order, non-mutually exclusive questions: “Are ‘unopened flowers’ present?,” “Are ‘open flowers’ present?,” “Are ‘fruits’ presents?” For gymnosperms the questions are: “Are ‘pollen cones’ present?” and “Are ‘seed cones’ present?” . The term “bud/s” can confuse floral buds with leaf buds; therefore, the PPO and this protocol refer to unopened flowers only. The second-order questions are not mutually exclusive. If unopened flowers, open flowers, and fruits are all present on a sheet, all questions can be answered in the affirmative. Having these data allows researchers to quickly identify the records that pertain to their individual research questions. The second-order questions require greater training for personnel to accurately discriminate unopened flowers, open flowers, and fruits. For many taxa , floral structures are small and distinguishing between unopened flowers, open flowers, and fruits can be challenging. Additionally, it is important that scorers are trained to distinguish between leaf buds and flower buds . As training materials are developed for various plant groups they should be shared widely across the community. While second-order scorings will determine which specimens should be included in phenological research, it is often valuable to know the specimen’s specific phenophase. Analyses can be more precise if we can distinguish between specimens in full flower from those specimens at the beginning or end of the flowering cycle. The third-order scorings are intended to place individual specimens at a specific point in phenological development. As such, these subcategories and the units used to report them may vary depending on the institutional or research priorities that generate them. We do not specify exactly what the third-order categories should be, as these will be determined by research priorities and staff time, but rather we explain how these questions are most commonly expressed or could be expressed within our proposed framework. Although we do not specify third-order categories, we do strongly recommend that researchers clearly define their categories and make their definitions broadly accessible, along with pertinent metadata. For example, the Simple Knowledge Organization System provides a framework for representing controlled vocabularies that easily lends itself to being shared online. The New England Vascular Plant project has devised a vocabulary following these guidelines and has published their vocabulary using SKOS . Furthermore, regardless of the nature of third-order categories, we strongly recommend that researchers share these data via the Darwin Core extensions explained below. For quantitative phenological data, some research groups require count data from a specimen, such as the numbers of unopened flowers, open flowers, and fruits on each sheet. Counts would be considered third-order scoring. For some analyses, raw count data may be transformed to express the proportions of reproductive organs represented by unopened flowers, open flowers, and fruits, thereby distinguishing specimens that represent early-, peak-, or late-flowering individuals. Even if count data are not precisely recorded from a sheet, the degree of flowering can be binned into categories representing early, peak, and late flowering . Note that in the example shown in Table 4, the third-order scorings, if categorical, are mutually exclusive. A sheet cannot simultaneously be ‘mostly unopened flowers’ and ‘mostly open flowers.’ Finally, in order to integrate third-order scorings with other sources of phenological data, we recommend use of the PPO.

A canonical prediction of economic theory is that high wages increase labor productivity. In settings where workers are salaried or paid by the hour, this is the concept of efficiency wages . In settings where workers are paid in proportion to their output , the theoretical connection between wages and productivity is even clearer. However, it has proven difficult to empirically estimate the responsiveness of labor productivity to piece rate wages, since much of these wages’ variation is driven by endogenous characteristics of the production process. In this paper, I provide the first quasiexperimental estimate of the elasticity of labor productivity with respect to piece rate wages. Specifically, I analyze a high-frequency panel of worker-level production data from over 2,000 California blueberry pickers paid by piece rates. Surprisingly, I find that on average, labor productivity is very inelastic with respect to wages. Piece rate wages are interesting to study because they offer such a direct, clear, and salient link between a worker’s effort and reward. In general, optimal labor contracts can be quite complex, as they must effectively incentivize worker effort while simultaneously accounting for issues like risk aversion, asymmetric information, and moral hazard . However, these complications are less of a concern in settings where a firm can cheaply monitor both worker productivity and product quality. In such cases, theory suggests piece rate wages will outperform other common incentive schemes . Understanding how workers respond to changes in a piece rate wage is important in sectors where these wages can vary over time, like in specialty agriculture, the auto repair industry, hydroponic net pots or the growing ride share market . Econometricians face a fundamental challenge when trying to estimate the causal effect of piece rate wages on labor productivity: these wages are inherently endogenous.

As an example, consider blueberry picking. When ripe berries are scarce and spread out , average worker productivity is low. When ripe berries are abundant and dense , it is easier for workers to pick berries quickly, and average productivity is markedly higher. Because farmers aim to keep their workers’ average effective hourly pay relatively stable over time, they set piece rate wages higher when picking is more difficult, and lower when picking is easier. In order to account for piece rates wages’ endogeneity, I adopt a two-pronged identification strategy. First, exploiting the richness of my multidimensional panel data, I econometrically control for environmental factors like seasonality and temperature that directly affect the berry picking production function. Second, I use the market price for blueberries as an instrument for piece rate wages. This price is a valid instrument because it affects a farmer’s willingness to raise piece rates , but is otherwise uncorrelated with picker productivity. Furthermore, the market price for California blueberries is set by global demand and global supply. As a result, individual farms are too small to directly affect the market price, and supply shocks at the farm level can be considered orthogonal to aggregate supply shocks. I find that, on average, labor productivity is very inelastic with respect to piece rate wages, and I can reject even modest elasticities of up to 0.7. This finding contrasts with both canonical economic theory and previous structural estimates: relying on a calibrated structural model of worker effort, Paarsch and Shearer estimate a labor effort elasticity of 2.14 in the British Columbia tree-planting industry, and Haley estimates a labor effort elasticity of 1.51 in the U.S. midwest logging industry. Why, then, do blueberry pickers not seem to respond to changes in their wage? One explanation of my findings could be that blueberry pickers respond to average effective hourly wages rather than marginal piece rate wages, similar to how electricity consumers respond to average prices rather than marginal prices . This is unlikely, both because piece rate wages are highly salient in the context I study, and because my identification strategy specifically isolates marginal effects from average effects.

Instead, I find suggestive evidence that blueberry pickers face some binding constraint on physical effort that is related to temperature. Specifically, I find that at moderate to hot temperatures, I cannot reject that the piece rate wage level has no effect on labor productivity. However, at temperatures below 60 degrees Fahrenheit , a one cent per pound increase in the piece rate wage increases worker productivity by nearly 0.3 pounds per hour, implying a positive and statistically significant productivity elasticity of approximately 1.6. In other words, blueberry pickers respond to the piece rate wage level at cool temperatures, but seem not to respond to changes in their wage at higher temperatures.Temperature also affects productivity directly in economically meaningful ways. Specifically, I find that blueberry pickers’ productivity drops precipitously at very hot temperatures: workers are 12% less productive at temperatures above 100 degrees Fahrenheit than they are at temperatures between 80 and 85 degrees Fahrenheit . However, I also find negative effects at cool temperatures. Workers are nearly 17% less productive at temperatures below 60 degrees Fahrenheit than at temperatures in the low eighties. The most likely explanation of this finding is that berry pickers lose finger dexterity at cool temperatures and find it uncomfortable to maintain high levels of productivity. This hypothesis is supported by evidence from the ergonomics literature , and highlights that temperature’s effects on labor productivity depend on the particularities of the relevant production process. To demonstrate the robustness of my findings, I address several threats to my identification strategy. First, I investigate berry pickers’ labor supply on both the intensive and extensive margins. I show that neither temperature nor wages have a statistically significant effect on these measures. Next, I address the fact that there exists a minimum hourly wage rule in the setting I study. This constraint binds for approximately 15.8% of my observations, raising concerns that workers falling below this threshold have an incentive to shirk or “slack off.” I re-estimate my results using only those observations where workers earn more than the minimum wage and see no qualitative change in my findings. Finally, I confront the possibility of adverse selection in my sample by limiting my sample to only the observations from workers who work more than thirty days in a single season. Previous studies have shown, and I confirm, that temperature affects labor productivity directly. However, I am the first to demonstrate that temperature also affects labor productivity indirectly by disrupting the economic relationship between wages and worker effort. As global temperatures rise, my findings suggest that firms in outdoor industries like agriculture and construction will have a reduced ability to effectively incentivize their employees’ productivity. This can have large economic consequences. In the $76 billion U.S. specialty crop sector, for instance, harvest labor can account for more than half a farm’s operating costs. This is also a setting where piece rate wages are common: in California alone, over 100,000 specialty crop farm workers were paid by piece rates in 2012. My econometric estimates allow me to make several predictions about how rising temperatures will affect the agricultural labor sector. To do this, I develop a model of a firm choosing an optimal piece rate wage under some exogenous environmental condition . My model produces two interesting sets of comparative statics. First, I show that temperature’s effect on the optimal piece rate wage depends on how temperature affects labor productivity directly, and how temperature affects labor productivity’s responsiveness to the wage. Plugging my empirical estimates into this model, I find that an optimizing blueberry farm would pay its workers a higher piece rate wage on particularly cool days, ceteris paribus. Second, I show that temperature’s effect on overall farm profits has the same sign as temperature’s direct effect on labor productivity.

In the case of California blueberry farms, blueberry grow pot where cool temperatures have meaningful negative effects on productivity, this suggests that the first-order effect of rising temperatures on profits is likely to be positive. However, in contexts where cool temperatures do not lower labor productivity, the opposite is likely to be true. The remainder of this paper is organized as follows: in section 1.2, I develop a simple theoretical model of workers’ optimal effort under a piece rate wage scheme. In section 1.3, I describe the institutional details of the two California blueberry farms I study in this paper. I then discuss my data and report summary statistics in section 1.4. Section 1.5 outlines my empirical strategy, and section 1.6 reports my results. I discuss my findings in section 1.7, giving particular attention to how rising temperatures are likely to affect the agricultural labor sector. Finally, in section 1.8, I conclude.There has been relatively little theoretical work done on piece rate wage schemes in the past, partly because their structure is so straightforward, and partly because they are so much less common than salaries or hourly wage schemes. Nonetheless, previous research has highlighted several important aspects of piece rate wages that are relevant to this paper. Prendergast and Brown both provide good summaries of when and where piece rates are likely to be effective. Specifically, in cases where firms can cheaply monitor productivity and ensure quality control, piece rates should correctly align workers’ incentives with those of their employer, maximizing labor productivity.10 Several papers have confirmed the prediction that, under the correct circumstances, piece rate wage schemes better incentivize labor productivity than do more traditional wage schemes. Lazear , studying an auto glass company, finds that a switch from hourly to piece rate wages boosts output per worker by an average of 44%. Shi , studying a tree-thinning company, estimates a more modest effect of 23%. Shearer , studying tree-planters in British Columbia, also finds an effect near 20%. Bandiera et al. study agricultural workers in the United Kingdom and come to a similar conclusion, noting that piece rates based on individual production eliminate cross-worker externalities found in relative incentive schemes. In a non-causal study from California, Billikopf and Norton also provide evidence that piece rate wages boost vine-pruners’ performance relative to hourly wages. Such increases in productivity under piece rates seem to come from increased worker effort, as Foster and Rosenzweig demonstrate by measuring workers’ net calorie expenditures under different pay schemes. None of the papers cited above, however, estimate how labor productivity responds to changes in a piece rate wage. Among the most well-known papers that have attempted to do so are Paarsch and Shearer and Haley . In both cases, the authors calibrate a structural model of worker effort in order to address piece rates’ endogeneity. They find positive elasticities of effort with respect to wages, of 2.14 and 1.51 respectively, in line with theoretical predictions. Other papers have relied on natural experiments or natural field experiments to try and recover the effect of piece rate wage levels on productivity. For instance, Treble exploits a natural experiment from the 1890s in an English coal mine to derive a near unit-elastic productivity response. In a more recent setting, Paarsch and Shearer implement a natural field experiment with tree-planters in British Columbia and estimate a productivity elasticity of 0.39. While the authors note that this estimate is “substantially smaller” than that of Paarsch and Shearer and Haley , it is unclear wether they think this result invalidates the earlier estimates. Finally, Guiteras and Jack conduct an experiment in rural Malawi to explore how variation in piece rate wages affects both quantity and quality of worker output. The authors find a positive but very inelastic effect of piece rate wages on workers’ output. Despite the theoretical simplicity of a piece rate wage scheme, it is not immune to employees’ behavioral responses. Even though a firm may be able to set a different piece rate every day, doing so may foment unrest among employees if the changes are seen as arbitrary . In other situations, high piece rates may operate as efficiency wages – à la Yellen , Shapiro and Stiglitz , and Newbery and Stiglitz – especially if a firm is trying to retain high-quality workers . An additional consideration is that variable piece rate wages may lead to a less reliable supply of labor on the intensive margin. In other words, piece rate employees may work fewer or more hours depending on the day’s wage. Such behavior would be consistent with a reference-dependent preference model like that of Kőszegi and Rabin where workers have some internal reference point for how much money they intend to earn in a particular day. Finally, piece rate wages are much more common in seasonal specialty agriculture than in many other industries or settings.

Assessing the relationship between the intake of nutrients and bio-actives from a whole food product to physiologic responses is difficult, as a multitude of processes are affected, including regulation of vascular function, provision of oxidant defense, and changes in gut microbiome profiles and subsequent output of secondary metabolites. Additionally, bio-actives from nuts and berries can interact with each other as well as other dietary components to alter bio-availability and health-promoting properties. For example, intake of dietary fats in conjunction with berries has been demonstrated to increase carotenoid bio-availability.Results could also be confounded by dietary changes made by participants in addition to incorporation of the test nut or berry. Habitual dietary intake is often measured through food frequency questionnaires or repeated 24-h dietary recalls. However, these subjective measures may not accurately capture the potential for nutrient-nutrient interactions that may alter polyphenolic or other bio-active components attributed to nut and berry consumption. Further complicating this issue is the observation that study designs utilizing longer-term interventions or that require the intake of a large amount of the test food are more likely to result in overreporting food intake due to fear that participants may be dismissed from the intervention. Most clinical trials using nuts and berries have been conducted in middle-aged or older Caucasian adults with one or more cardiometabolic risk factors.

Whether these results extend to other population groups is either inferred or unknown. Future research would benefit from extending the study populations to include those from other racial and ethnic groups. This is particularly important inorder to address the current NIH research initiative in precision nutrition and health, dutch bucket hydroponic the “Nutrition for Precision Health powered by the All of Us Research Program”. The inclusion of biological females in clinical nutrition trials is imperative, yet the current literature includes predominantly male participants. Because many studies on nuts and berries focus on cardiometabolic outcomes, the unique aspects of female physiology must be considered. For example, vascular function fluctuates with the phase of the menstrual cycle, which has largely been ignored in most past studies. More studies are also needed in young children as well as in young adults up to about the age of 40. A pilot study reported a correlation between blueberry supplementation and acute positive effects on memory and executive function in 7- to 10-y old children. A large study among pregnant women-infant dyads reported positive protective neuropsychological effects on long-term cognitive development in children at 1, 5, and 8 y of age when nuts were consumed during gestation. Finally, translation of research results is challenging when considering socioeconomic status , particularly when food items are not accessible or affordable. Barriers to participation in clinical research studies among those of low SES include a low interest in clinical trials, inefficient or inadequate explanation of the study in culturally appropriate terms, participants’ distrust of biomedical research, and participant burden, including lack of transportation or the inability to prioritize participation in research over work obligations.

Like many other dietary studies, research on nuts and berry studies often use acute studies evaluating postprandial effects. However, either a lack of or successful demonstration of benefits does not necessarily predict a similar outcome over extended periods of intake. Depending on the outcome measure, detectable effects may take weeks or months for the intervention. Only a limited number of studies exist assessing the impact of nut or berry intake on the incidence or severity of diseases or metabolic dysfunction, which require durations of months or years.Precision nutrition evaluates an individual’s unique biological characteristics such as genotype and phenotype, including DNA expression, influences of the gut microbiome, and metabolic response to specific foods or dietary patterns, as well as dietary habits and external factors influencing outcomes such as social determinants of health, to determine the most effective dietary strategies to improve health and prevent disease. Understanding the sources of interindividual variability that contribute to metabolic heterogeneity and applying mathematical modeling and computational algorithms will be essential to refining dietary recommendations. Several recent publications comprehensively review research gaps and study design considerations in the field of precision nutrition and specifically concerning phenolic-rich plant foods. Precision nutrition will lead to important discoveries pertaining to interindividual responsiveness to the intake of nuts and berries. Ultimately, this information can be applied via targeted recommendations to individuals and groups for achievable and sustainable dietary intake of nuts and berries to promote optimal health. The incorporation of bio-monitoring technologies into study designs may also be used for precision nutrition.

Current and emerging mobile devices can provide continuous data collection in free-living populations with minimal participant burden. The study of nuts and berries would be enhanced with the use of devices that can capture real-time physiological outputs at home that reflect normal living conditions. Further collaborative efforts in the fields of bioengineering and artificial intelligence hold promise for advancing the understanding of benefits from nuts or berries. An emerging personal bio-monitoring technology is the Precision Health Toilet, which collects and evaluates human urine and stool, which are then analyzed using artificial intelligence to determine flow rate and volume of urine, as well as fecal analysis via the Bristol Stool Scale. A second type of toilet seat, the Heart Seat, has recently been approved by the US Food and Drug Administration for home use to monitor heart rate and oxygen saturation, with future plans to add sensors that monitor systolic and diastolic blood pressure. Assessment of metabolites in the excreta seems like a feasible goal for future development, which may be useful, for example in the detection of urinary and fecal metabolites that can reflect the metabolism of ellagic acid to urolithins and of -epicatechin to γ-valerolactone. A third example is an ingestible capsule containing a biological photosensor that can detect gut inflammation. Bioluminescence can be monitored from bacteria that have been engineered to illuminate when they come into contact with a molecule for which they have been coded, such as urolithins from berries or lipid-sensitive metabolites from nuts. Finally, another type of ingestible capsule has recently been detailed that collects samples from multiple regions of the human intestinal tract during normal digestion. This device has been used to explore the role of the gut microbiome in physiology and disease, with novel findings that intestinal and stool metabolomes differ dramatically. The ability of nut or berry intake to alter such metabolomes, and their association with changes in physiological function and health outcomes, would be an interesting area for future research. Although these technologies are still in their infancy, they have promise to further precision nutrition research efforts on nuts and berries. Research addressing the issue of “responders” compared with “non-responders” is important in understanding the metabolic discrepancies in many studies on nuts and berries. For example, platelet aggregation phenotypes can vary significantly by individual responsiveness to oxylipins, bio-active lipid mediators derived from polyunsaturated fatty acids present in nuts as well as in extra virgin olive oil. Variations in circulating metabolites and microvascular function following the intake of freeze-dried strawberry powder have been reported. Those individuals producing increased nitrate and nitrite levels showed favorable changes in function whereas those showing no change in nitrate or nitrite levels did not. Another example is illustrated by a recent letter in response to a systematic review of almond intake and inflammatory biomarkers. The letter notes that while the review included amounts of almonds ranging from 10 to 113 g/d, favorable responses only occurred at intake of <60 g/d. Further, the authors note that although the review reports beneficial effects of almond intake on reduction in C-reactive protein and interleukin-6, subgroup analyses showed that the effects on these 2 outcomes were not significant among those with obesity or who were rated as unhealthy prior to the intervention. Characterizing participants according to precision nutrition, including the use of genetic phenotyping to identify target genes that may result in “responders” and “non-responders” prior to enrollment may be helpful for clinical trials but does not reflect responses in a free-living population.

Furthermore, dutch buckets system in addition to physiological variations, sociobehavioral differences among individuals that may modulate responses to berries and nuts must also considered. Nonetheless, innovative precision nutrition models that can identify interindividual differences would be useful in defining mechanisms of action and potentially who would benefit the most from regular nut or berry consumption. Plasma and serum concentrations are useful to identify the bio-availability and bio-efficacy of key nutrients and phytochemicals found in nuts and berries. Some compounds, such as small molecular weight polyphenols, are first absorbed in their native state in the small intestine. Other polyphenols can be biotransformed via the host microbiota to a second set of compounds that are subsequently absorbed and confer additional bio-activity beyond that obtained from the parent molecules. Monitoring both host and microbial metabolites in the blood and urine, and those that may accumulate in tissues of interest such as the liver and gastrointestinal epithelium, among other tissues, would be useful in understanding the dynamics of nut and berry bio-activity and specific association with site of actions. Broader application of orthogonal approaches that combine untargeted with targeted metabolomic platforms and combined with the use of advanced informatics will support new understanding about the absorption, distribution, metabolism, and excretion of compounds found in nuts and berries. For example, the UC Davis West Coast Metabolomics Center conducts both targeted and untargeted assays that assess plasma microbial metabolites using a biogenic amine panel that identifies and quantifies acylcarnitines, trimethylamine N-oxide, cholines, betaines, nucleotides and nucleosides, methylated and acetylated amines, di- and oligo-peptides, and a number of microbially modified food-derived metabolites. Some interindividual differences in response to nut or berry intake have been attributed to the composition of the gut microbiome. For example, ellagitannins are polyphenolic compounds present in strawberries, raspberries, and walnuts that are metabolized by gut bacteria into an array of urolithins. The production of urolithins relies on the capacity of specific microbes, Gordonibacter pamelaeae and Gordonibacterurolithinfaciens. Urolithins may decrease symptoms of chronic metabolic diseases, including inflammation and dyslipidemia. Following a single intake of red raspberries, individuals with prediabetes and insulin resistance had lower concentrations of circulating urolithins compared to levels found in those who were metabolically healthy, a result related to gut microbiome composition. In the same population, consuming red raspberries for 4 wk improved hepatic insulin resistance and total and LDL cholesterol in the prediabetes group, and the effects were related to decreased R. gnavus and increased E. eligins. Overall, including a practical amount of red raspberry in the diet regularly is a low-calorie dietary strategy that improves gut microbiota composition and function in individuals with prediabetes and insulin resistance resulting in improvements in metabolic health. With a sustained emphasis on the role of gut microbiota in nutrition research, advances in our understanding of food-gut dynamics will provide new insights about the role of nuts and berries in human health and performance. Although research on a specific nut or berry provides insight into bio-activity and potential mechanisms of action, such focus also creates the potential for fragmentation because the search for overall dietary patterns is not addressed. The composition of fruits and nuts differ at the molecular level, and a broader view assessing similarities in chemistry and health benefits is critical for translational research as well as for messaging purposes. For example, blueberries, strawberries, pomegranate, walnuts, and grapes all have reported benefits for cardiovascular health, driven largely by the presence of similar polyphenols, which are present at varying quantities in each of these foods. Although health professionals and consumers often hear messaging on a single berry or nut, the potential benefits of increasing consumption of the broader category may be obscured or lost. This challenges the ability to maintain consistent messaging and align better with translatable dietary guidance. Future interventions that combine nuts and berries with one or more other foods within a food matrix at dietary achievable doses and in more diverse populations are warranted. To date, multi-omics technologies have provided valuable insights into exposure-disease relationships. Coupled with artificial intelligence, predictive modeling and continuous, personalized monitoring, these data-intensive outcomes can provide further insights about the health benefits associated with regular intake of nuts or berries. Use of highly personalized data collection devices will require secure data repositories.

With an increasing global population, new tools are needed to provide highly nutritive foods and low-cost therapeutics to a larger populace. Synthetic biology is a proven approach to reconstitute metabolic pathways for the production of valuable chemicals; so far, microbes have been the production platform of choice. While an extremely robust synthetic biology platform, microbes are unicellular organisms, hindering the reconstitution of certain complex plant metabolic pathways that normally take place in multiple cellular compartments and tissue types . Additionally, many plant enzymes do not function properly in microbial systems . Microbial production also requires the use of expensive, large-scale fermenters and the extraction of metabolites produced, incurring significant costs. Plants are ideally suited for the production of plant natural products as they natively host many of the biosynthetic pathways and produce essential precursor molecules required using light and water. Additionally, plants have a variety of tissues and organelles, allowing for compartmentalization of pathways and toxic intermediates. As humanity’s main source of food, plants are a unique yet practical vehicle for the production and delivery of essential nutrients and nutraceuticals . While plants present an ideal platform, there are still technological bottlenecks hampering our ability to optimize the production and accumulation of specific nutrients and phytochemicals. This review will examine the past successes and future challenges of plant synthetic biology in producing valuable therapeutics and enhancing the nutritive capacity of food crops. Additionally, strawberry gutter system the strengths and weaknesses of plants as a production platform will be addressed.

Plants and the compounds they produce are some of the most important natural resources we have. From the food we eat to many of the medicines we take, plants provide us with a diverse selection of bio-active compounds capable of improving human health. A major goal of plant synthetic biology is to identify biosynthetic pathways for bio-active compounds and transfer them to other plant species to provide alternative sources of these compounds. In this section, we focus on the progress of pharmaceutical compound biosynthetic pathway discovery, efforts for enhancing the nutritive value of plants, and exploration of nutraceuticals for better health and wellness.Plant remedies have provided insight into functional molecules that have drastically improved human healthcare, such as the revolutionization in pain management following the use of opiates from opium poppy and the discovery of the active ingredient, salicin, in willow bark which enabled the synthesis of aspirin by the acetylation of plant derived salicylic acid . However, as was the case with aspirin, it can be difficult to pinpoint the specific metabolite, or combination of metabolites, that result in observed beneficial effects from the entirety of a plant’s metabolome. With the major advances in modern analytical chemistry tools, it has become easier to identify and characterize specific metabolites from any given organism . From this collection of metabolites, specific compounds can be isolated and tested for bioactivity and efficacy against many chronic and deadly diseases. However, knowing the identity of the compound is only the start, as many of these active compounds are found in uncultivated plant varieties or in low abundance therein, impeding the large-scale production necessary for pharmaceutical use . Plant synthetic biology seeks to circumvent some of these problems through heterologous expression of bio-active compounds for large-scale production and extraction.

The first step needed to achieve this goal is to fully characterize the biosynthetic pathway for the compound of interest. Understanding the reactions between pathway intermediates and determining the required parts in a pathway based on standard classes of enzymes can help to fill in the gaps of many incomplete biosynthetic pathways . Additionally, modern sequencing technologies have advanced to the point where whole genome sequencing and transcriptomics in conjunction with co-expression analysis, phylogenomics, gene clustering, and genome-wide association studies of non-model species have become accessible . These techniques enable a route to the rapid discovery of all the parts in complex biosynthetic pathways, which enables heterologous expression of pathways for compound production. A notable example of this is the discovery of the pathway for the anti-inflammatory drug, colchicine, from Gloriosa superba . In this work, the authors used previously generated RNA-seq data from multiple species, generated a new RNA-seq dataset, and used metabolomics to determine eight essential genes in the pathway as well as reconstitute a 16-gene pathway with transient expression in Nicotiana benthamiana to produce the colchicine precursor, N-formyldemecolcine. With these tools in hand, it is time for plant synthetic biology to develop a library of parts and pathways to not only produce natural bio-active compounds for better human health but also new-to-nature compounds which could revolutionize the way we think about wellness. Other examples of heterologous expression of phytochemical pathways have shown it is possible to develop plants into pharmaceutical production platforms.

Crocosmia spp. is an ornamental plant that was found to produce Montbretin A , a potent inhibitor of human pancreatic amylase, which is a promising treatment for type II diabetes awaiting clinical validation . However, MbA is only produced in the corms of Croscosmia spp. in low amounts. Corms are the small vegetative reproductive tissue of the plant, making the propagation and extraction of MbA difficult. Thus, the limitations of Crocosimia spp. make a heterologous host a better suited system for MbA production. The full MbA pathway was recently discovered and its heterologous expression in leaf tissue of N. benthamiana resulted in a measurable but low yield, displaying the need for pathway and potentially host optimization for largescale production. Potential mechanisms for effective pathway optimization include enhancing precursor molecule levels or relaxation of bottleneck steps within the pathway. For example, etoposide is a chemotherapy drug whose precursor previously could only be isolated from the endangered mayapple plant, Sinopodophyllum hexandrum. Optimization of the production of the etoposide precursor, -deoxypodophyllotoxin, was improved by two orders of magnitude through transient expression of eight genes required to produce coniferyl alcohol, allowing for purification of milligram levels . Traditional medicinal plants are another source of pharmacologically important compounds, with several compounds having fully elucidated biosynthetic pathways. Salidroside, isolated from the Rhodiola genus, has a long history of use due to its potential, albeit understudied, benefits for mood stabilization, fatigue, and prevention of cardiovascular disease and cancer . Unfortunately, because of its suggested benefits, some species of Rhodiola are being harvested to near extinction from their native habitats. The recent discovery of its three-enzyme biosynthetic pathway using transcriptomics and metabolomics has enabled the production of salidroside by heterologous expression in N. benthamiana, providing a scalable alternative to extraction from Rhodiola . From another medicinal plant, Catharanthus roseus, the final steps of the complicated, thirtyone enzyme biosynthetic pathway of vinblastine, an important drug in the treatment of leukemia and lymphoma, have been discovered using RNA-seq to identify the genes and transient expression in N. benthamiana to validate vinblastine production . The heterologous expression of this pathway could provide an alternative production platform for the synthesis of this important pharmaceutical. Even with these discoveries, there are many biosynthetic pathways of important medicinal compounds that are not fully understood, such as tentative antivirals found in traditional Chinese medicine . Currently, some of these traditional medicines are undergoing FDA clinical trials to be developed into direct treatment or combinatorial supplements with existing approved drug treatments . With continued progress, grow strawberry in containers there will be a more comprehensive library of pharmacologically relevant phytochemical pathways to draw upon for the treatment of any type of disease. With a greater understanding of the biosynthetic principles behind natural product synthesis, synthetic biologists are producing new-to-nature molecules by utilizing well characterized precursors in combination with modifying enzymes from multiple plant sources. Terpenoids are ubiquitous across the plant kingdom and this diverse class of molecules serves many functions for plants, including but not limited to defense and stress response.

Researchers have demonstrated that heterologous expression of the triterpene, ß-amyrin, with combinations of CYP450 enzymes can produce more than a dozen natural saponins as well as new-to-nature products on a gram scale . These compounds were tested for anti-proliferative and anti-inflammatory effects and demonstrated some effectiveness, illustrating the potential pharmacological efficacy of new-to-nature compounds being produced in plants at a large scale. Other evidence shows that the addition of plant and bacterial enzymes into a plant-specific pathway can also enhance the ability of plants to generate new to-nature compounds. A recent example of this is the production of novel crucifalexins and halogenated derivatives of brassinin by transiently expressing halogenases and various CYP79 genes with different specificities . The authors also demonstrated that the anti-fungal nature of these new compounds was enhanced compared to commercially available products, showing an alternative approach to developing a suite of new compounds using the parts and pathways that are readily available.One of the primary ways plant synthetic biology can aid in health and wellness is by alleviating malnutrition which is still prevalent across the entire world. Beyond the macromolecular necessities of food, an even greater portion of the world experiences micronutrient deficiencies—termed “hidden hunger”– especially in regard to vitamin A, iodine, zinc, and iron . A recent review has an in depth analysis of the types of deficiencies as well as some strategies that have been used to biofortify crops. The production of palatable crops that provide adequate amounts of macroand micronutrients that can grow in diverse biomes is one of the cheapest and most practical ways to ameliorate malnutrition . Plant metabolic engineering has the potential to address many of these nutrient shortcomings through both the manipulation of existing pathways and the introduction of additional plant pathways. Traditionally the enhancement of crop species has occurred through breeding practices which involves a series of genetic crosses and backcrosses to take a desirable trait from one variety of plant into a commercial cultivar. This process requires multiple plant generations and can introduce undesirable effects due to tight gene linkage. These practices have been in use for thousands of years and have improved almost every crop that we eat today, yet the arduous nature of plant breeding has limited the varieties of crops that are actively cultivated for consumption. With the advent of gene-editing technologies, scientists can directly modify specific DNA sequences to produce desired changes in the plant genome within one generation. An example of this is in the improvement of ground cherry, a species closely related to tomato . In this work, the authors use the clustered regularly interspaced short palindromic repeats –CRISPR-associated protein-9 nuclease system to generate null mutations in Ppr-SP5G and SlCLV3 promoter region to impart desirable traits into this crop, such as flowering and fruit size. Additional examples of de novo domestication from wild tomatoes have shown the potential of nutritive enhancement along with cultivation improvement, retaining the disease and abiotic stress resistance of the wild varieties . With the use of new tools to perform genetic modifications, such as CRISPRCas9, species of plants that are difficult to cultivate can be modified for use as crops, thereby diversifying available food options. This could in turn enhance the availability of nutrients that are limited throughout the world. Additionally, the advent of a CRISPR-Cas9 with less restriction on the genetic site of protein binding will allow for greater flexibility in the use of this tool . Gene editing further allows for the alteration and enhancement of specific endogenous biosynthetic pathways to produce biofortified crop species. Work has shown that silencing of lycopene ε-cyclase in the carotenoid pathway can enhance the natural levels of ß-carotene while reducing the α-carotene levels . Other examples show that the manipulation of plastid identity—such as the conversion of chloroplast to chromoplast—can enhance the overall carotenoid levels. One study converted chloroplasts to chromoplasts in tomato during early fruit development through the ectopic expression of the Arabidopsis thaliana ORANGE gene containing a SNP . Another study converted chloroplasts to chromoplasts throughout the plant through the expression of plastid-targeted microbial phytoene synthase in multiple plant species; however, the transgenic plants suffered from decreased photosynthetic efficiency in leaf tissue . This highlights the importance of finding the balance between high titers and stable growth. Additionally, the levels of zeaxanthin, a carotenoid with potential benefits in eye health, have been increased by the elimination of zeaxanthin deepoxidase in the algal species Chlamydomonas reinhardtii . The enhancement of desirable carotenoids could easily be translated into higher plant species and much effort is being made for their use in non-model organisms .

Superior fruit quality is also associated with sugar levels. During fruit ripening, sugar levels of the endocarp increase by importing hexose symplastically and/or apoplastically. Sugar transporters , sucrose transporter, and tonoplast sugar transporter have been demonstrated to regulate intercellular sugar transport in phloem and fruit.To the best of our knowledge, we are the first to report on the potential role of these genes during blueberry fruit development. In addition, homologs of A. thaliana TST1 and watermelon ClTST1 and ClTST3 were expressed during fruit ripening in blueberry. Elevated expression of a ClTST1 homolog was observed throughout fruit development, but the ClTST3 homolog showed very low expression. Another gene that is highly expressed during fruit maturation is vacuolar invertase. As described in other systems, its upregulation during fruit ripening coincided with the breakdown of starch to sucrose or a mixture of glucose and fructose, suggesting that it may be involved in the regulation of sugar accumulation in blueberry fruit. It was previously reported that vacuolar invertase modulates the hexose to sucrose ratio in ripening fruit. In addition, there are also two sugar transport protein homologs that exhibited developmental specific expression. However, their function remains largely unknown, thus, their potential role in sugar accumulation in the developing berry requires further investigation.Tandemly duplicated genes arise as a result of unequal crossing over or template slippage during DNA repair, exhibit high birth-death rates, grow bucket and typically are in co-regulated clusters in the genome.

Smaller scale duplications, which include tandem duplicates, are highly biased toward certain gene families including those involved in specialized metabolism. Furthermore, tandem duplications often results in the increased dosage of gene products and may improve the metabolic flux of ratelimiting steps in certain bio-synthetic pathways. Most genes associated with the biosynthesis of antioxidants have at least one tandem duplicate present in the highbush blueberry genome, with tandem array sizes ranging from 2 to 10 gene copies . The largest tandem arrays were found for HQT and HCT genes, which are co-regulated and involved in the CGA pathway . Differences in tandem array sizes were also observed between homoeologous chromosomes for various genes. For example, the C3H gene, which is involved in CGA biosynthesis , was present on all four homoeologous chromosomes but with varying tandem array sizes. One of the homoeologous chromosomes had two copies of C3H, while the other three homoeologous chromosomes had four copies. This suggests that copy number differences of C3H among subgenomes may be due to either selection for gene duplication or loss or, in the case of allopolyploidy, may be due to preexisting gene content differences among the diploid progenitor species. Genes in the anthocyanin pathway with other unique duplication patterns include CHS, CHI, OMT, and UFGT. The gene CHS, involved in the conversion of 4-coumaryl-CoA to naringenin chalcone, has two copies, and both have tandem duplicates in at least three of the homoeologous chromosomes. Interestingly, the gene CHI has a single preserved tandem gene duplicate on only one of the homoeologous chromosomes. However, additional copies of CHI were also identified more distantly away from the syntenic ortholog on another homoeologous chromosome, likely involving a transposition event following tandem duplication.

The OMT and UFGT genes all have tandem duplicates on all of the homoeologous chromosomes, although with varying array sizes, while the ANR gene involved in the conversion of anthocyanidin to proanthocyanidin is single copy on all homoeologous chromosomes. DFR gene, which is involved inthe conversion of dihydroquercetin/dihyromyricetin to leucoanthocyanidin, has a single tandem duplicate on only one of the homoeologous chromosomes. These findings suggest that there may have been greater selective pressure to retain tandem duplicates for genes encoding enzymes involved in anthocyanin production than conversion to proanthocyanidins. The vast majority of tandem duplicates are eventually lost ; however, in rare instances, some may undergo functional diversification. Gene expression analysis revealed that 83.4% of the tandem duplicates were expressed in at least one transcriptome library with 73.5% expressed in at least one of the fruit developmental stages. This suggests that a subset of these duplicate genes have non-functionalized, subfunctionalized, or neofunctionalized. Future studies are needed to more thoroughly investigate the functions of these genes with more diverse libraries and additional transcriptome analyses.Despite the economic importance of blueberry, molecular breeding approaches to produce superior cultivars have been greatly hampered by inadequate genomic resources and a limited understanding of the underlying genetics encoding important traits.

This has resulted in breeders having to solely rely on traditional approaches to generate new cultivars, each with widely varying fruit quality characteristics. For example, our analysis of a diversity panel consisting of 84 cultivars and wild species revealed that ”Draper” has antioxidant levels that are up to 19x higher than other cultivars. Thus, the genome of ”Draper” should serve as a powerful resource to the blueberry community for guiding future breeding efforts aimed at improving antioxidant levels among other important fruit quality traits. Furthermore, to our knowledge, this is not only the first genome assembly of the cultivated highbush blueberry but is also the first chromosome-scale and haplotype-phased genome for any species in the order Ericales. Ericales includes several other high-value crops and wild species with unique life history traits . Thus, we anticipate that this reference genome, plus associated datasets, will be useful for a wide variety of evolutionary studies. Here, we also leveraged the genome to identify candidate genes and pathways that encode superior fruit quality in blueberry, including those associated with pigmentation, sugar, and antioxidant levels. Furthermore, we found that genes encoding key biosynthetic steps in various antioxidant pathways are enriched with tandem gene duplicates. For example, tandem gene duplications have expanded gene families that are involved in the biosynthesis of anthocyanins. This suggests that, in addition to a recent whole genome duplication, tandem duplications may have greatly contributed to the metabolic diversity observed in blueberry . These tandem duplicates may have evolved new functions , possibly involved in the biosynthesis of novel compounds, and/or were selected to improve the metabolic flux of specific biosynthetic steps that alter the dosage of certain endpoint metabolites. Future studies are needed to further investigate the possible role of tandem duplications in having modified metabolite levels and composition in wild and cultivated blueberry. Our analyses also revealed that highbush blueberry, a tetraploid, likely arose from the hybridization of two distinct parents, possibly allopolyploidy, based on the sequence divergence, unique transposable element insertions, and subgenome expression patterns. Our analyses revealed that the subgenomes in highbush blueberry may be controlling a distinct set of genetic programs . The dominantly expressed subgenome in most surveyed tissues becomes the lowest expressed during fruit development. This observation is similar to findings in allopolyploid wheat where developmental and adaptive traits were shown to be controlled by different subgenomes. For example, cell type- and stage dependent subgenome expression dominance was observed in the developing wheat grain. We argue that both highbush blueberry and hexaploid wheat, each now with high-quality reference genomes, make excellent systems to further investigate these underlying mechanisms of subgenome dominance. Subgenome dominance has far-reaching implications to numerous research areas including breeding efforts. For example, marker-assisted breeding needs to target the correct set of dominant homoeologs given the trait in polyploids that exhibit subgenome dominance. Thus, we anticipate that this genome, combined with improved insights into subgenome dominance, dutch bucket for tomatoes will greatly accelerate molecular breeding efforts in the cultivated highbush blueberry.The genome of ”Draper” was assembled using the DeNovoMAGIC software platform , which is a de Bruijn graph-based assembler designed for higher polyploid, heterozygous, and/or repetitive genomes.

The Chromium 10X data were utilized to phase, elongate, and validate haplotype scaffolds. Four Dovetail Hi-C libraries were prepared as described previously and sequenced on an Illumina HiSeq X system with paired-end 150 bp reads to a total of 90.7X physical coverage of the genome . The de novo genome assembly, raw genomic reads, and Dovetail Hi-C library reads were used as input data for HiRise, a software pipeline designed specifically for using proximity ligation data to scaffold genome assemblies. Illumina genomic and Dovetail Hi-C library sequences were aligned to the draft input assembly using a modified SNAP read mapper. The separations of Dovetail Hi-C read pairs mapped within draft scaffolds were analyzed by HiRise to produce a likelihood model for genomic distance between read pairs, and the model was used to identify and break putative misjoins and to make joins to close gaps between contigs.Plant tissue samples were collected from blueberry cv. Draper grown in the growth chamber . For the fruit developmental series, three biological replicates each of berries at seven developmental stages were collected from cv. Draper in a field at the Horticulture Teaching and Research Center, Michigan State University, in July 2017. All plant tissues were immediately flash frozen in liquid nitrogen, and total RNA isolation was performed using the KingFisher Pure RNA Plant kit . Isolated total RNA was quantified using a Qubit 3 fluorometer . All samples were submitted to the Michigan State University Research Technology Support Facility Genomics core and sequenced with paired-end 150 bp reads on an Illumina HiSeq 4000 system .The draft genome of V. corymbosum cv. Draper was annotated using the MAKER annotation pipeline. Transcript and protein evidence used in the annotation included protein sequences downloaded from A. thaliana and UniprotKB plant databases, V. corymbosum ESTs from NCBI, and transciptome data assembled with StringTie from different blueberry tissues . A custom repeat library and Repbase were used to mask repetitive regions in the genome using Repeatmasker. Ab initio gene prediction was performed using gene predictors SNAP and Augustus. The resulting MAKER Max gene set was filtered to select gene models with Pfam domain and annotation edit distance <1.0. The filtered gene set was further scanned for transposase coding regions. The amino acid sequence of predicted genes was searched against a transposase database. The alignment between the genes and the transposases was further filtered for those caused by the presence of sequences with low complexity. The total length of genes matching transposases was calculated based on the output from the search. If more than 30% of gene length aligned to the transposases, the gene was removed from the gene set. Furthermore, to assess the completeness of annotation, the V. corymbosum Maker standard gene set was searched against the BUSCO v.3 plant dataset . Genes were annotated with pfam domains using InterProScan v5.26–65.0.To identify and classify repetitive elements in the genome, LTR retrotransposon candidates were searched using LTRharvest and LTR finder and further identified and classified using LTR retriever . A nonredundant LTR library was also produced by LTR retriever. Miniature inverted transposable elements were identified using MITE-Hunter. MITEs were manually checked for target site duplications and terminal inverted repeats and classified into superfamilies . Those with ambiguous Target Site Duplication and Terminal Inverted Repeats were classified as ”unknowns.” Using the MITE and LTR libraries, the V. corymbosum genome was masked using Repeatmasker. The masked genome was further mined for repetitive elements using Repeatmodeler. The repeats were then categorized into two groups: sequences with and without identities. Those without identities were searched against the transposase database; if they had a match, they were considered a transposon. The repeats were then filtered to exclude gene fragments using ProtExcluder and summarized using the ‘fam coverage.pl’ script in the LTR retriever package. The assembly continuity of repeat space was assessed using the LLAI deployed in the LTR retriever package. LAI was calculated based on either 3 Mb sliding windows or the whole assembly using LAI = /Total LTR-RT length. For the sliding window estimation, a step of 300 Kb was used . To account for dynamics of LTR retrotransposons, LAI was adjusted by the mean identity of LTR sequences in the genome based on all-versus-all blastn search, which was also performed by the LAI program.Illumina adapters were removed from the raw reads using Trimmomatic/0.33, and trimmed reads were filtered using FASTX Toolkit. After quality assessment using FastQC , the filtered reads were then aligned to the V. corymbosum genome using STAR. For the samples that were used for annotation, transcript assembly was performed de novo using StringTie.

These findings also will help breeders to select for genomic variants underlying volatiles important to flavor. New markers can be designed from regulatory regions of key aroma volatiles, including multiple medium-chain volatiles shown to improve strawberry flavor and consumer liking , methyl thioacetate contributing to overripe flavor and methyl anthranilate imparting grape flavor . In the present study, a new functional HRM marker for mesifurane was developed and tested in multiple populations . These favorable alleles of volatiles can be pyramided to improve overall fruit flavor via marker assisted selection. Strawberry also shares common volatiles with a variety of fruit crops. Specific esters are shared with apple , certain lactones are shared with peach and various terpenes are shared with citrus . Syntenic regions and orthologous genes could be exploited for flavor improvement in those species. Additional insights were gained for the strawberry gene regulatory landscape, SV diversity, complex interplays among cis- and trans- regulatory elements, and subgenome dominance. Previously, Hardigan et al. and Pincot et al. showed a large genetic diversity existing in breeding populations of Fragaria × ananassa, challenging previous assumptions that cultivated strawberry lacked nucleotide variation owing to the nature of its interspecific origin and short history of domestication . Our work corroborated their findings and showed that even highly domesticated populations harbor substantial expression regulatory elements and structural variants. Over half of the expressed genes in fruit harbored at least one eQTL, and 22 731 eGenes had impactful cis-eQTL. The distribution of trans-eQTL is not random, hydroponic nft channel but rather is concentrated at a few hotspots controlled by putative master regulators .

The aggregation of trans-eQTL also was observed in plant species such as Lactuca sativa and Zea mays . Furthermore, we observed a substantial number of trans-eQTL among homoeologous chromosomes, similar to observations in other allopolyploid plant species . In cotton, physical interactions among chromatins from different subgenomes have been identified via Hi-C sequencing , supporting a potential regulatory mechanism among homoeologous chromosomes. However, owing to the high similarity among four subgenomes and limited length of Illumina reads, false alignment to incorrect homoeologous chromosomes could arise, leading to ‘ghost’ trans-eQTL signals. Future studies are needed to scrutinize the homoeologous trans-eQTL and investigate the mechanism behind this genome-wide phenomenon. Higher numbers of trans-eQTL in the Fragaria vesca-like subgenome are consistent with its dominance in octoploid strawberry . By contrast, the highly mixed Fragaria viridis- and Fragaria nipponica- like subgenomes contained much smaller numbers of trans-eQTL. The characterization of naturally-occurring allelic variants underlying volatile abundance has direct breeding applications. First, this will facilitate the selection of desirable alleles via DNA markers. Second, understanding the causal mutations in alleles can guide precision breeding approaches such as gene editing to modify the alleles themselves and/or their level of expression. From a broader perspective, multi-omics resources such as this one will have value for breeding a wide array of fruit traits. Enhancing consumer satisfaction in fruit ultimately will depend on the improvement of the many traits that together enhance the overall eating experience.

Natural competence is a phenomenon that allows bacteria to take up DNA segments from the environment and incorporate them into the genome via homologous recombination . Natural competence was first demonstrated in Streptococcus pneumoniae in 1928 by Frederick Griffith . Griffith showed that virulence genes were transferred from donor to recipient cells, converting the nonvirulent recipients into virulent pathogens . Since then, 80 bacterial species in divergent phyla have been described as naturally competent . Although the exact reasons for occurrence of natural competence in bacteria still remain unknown, studies showed that natural competence is induced under conditions of starvation and DNA damage , and it has been hypothesized that the incoming DNA serves as a food source and DNA repair material. Another proposition is that natural competence allows acquisition of new genes and alleles, providing the recipient cells with adaptive advantages. In fact, a previous study showed an increased rate of adaptation by natural competence in Helicobacter pylori . Interestingly, natural competence has been demonstrated in some of the most highly diverse and successful human pathogens such as H. pylori , Neisseria meningitidis and Neisseria gonorrhoeae , and Porphyromonas gingivalis , which require rapid adaptation to evade the immune response. Furthermore, natural competence also was described in two plant pathogens, Ralstonia solanacearum andXylella fastidiosa , both of which have very broad plant host ranges. Xylella fastidiosa is a bacterial pathogen affecting many economically important crops, such as grape, citrus, coffee, peach, and almond . The disease process is not completely understood, but it is proposed that X. fastidiosa forms biofilm-like aggregates and blocks xylem vessels, the conduits for water and nutrient transport in the plants . This blockage hinders xylem sap flow and starves the upper aerial parts of water and mineral nutrients, producing symptoms that resemble those of water and nutrient deficits. X. fastidiosa is transmitted by a number of xylem sap-feeding insects, including sharpshooter leaf hoppers and spittlebugs in which X. fastidiosa forms biofilms in the foregut . Taxonomically, X. fastidiosa is divided into five subspecies based on multilocus sequence typing . Even within the subspecies, host range and genotype diversity have been described , and recombination events among strains have been detected among field-collected samples . In fact, homologous recombination was shown to have a greater effect in generating genetic diversity in X. fastidiosa than point mutation . Recent outbreaks of X. fastidiosa diseases in Europe and Asia and also in new plant hosts such as olive , blueberry , and pear suggest the great adaptation potential of this pathogen. In a number of plant species, X. fastidiosa is believed to live as a harmless endophyte without inducing disease symptoms . Coexistence in the same xylem system of different strains for a long time without killing the host represents a fertile environment for exchange of DNA material. Several MLST-based studies detected intersubspecific recombination among strains of X. fastidiosa and proposed recombination as the mechanism of new allele acquisition, leading to plant host shift and disease emergence. Intersubspecific recombination was described to generate strains that infect citrus and coffee , mulberry , and blueberry and blackberry . A recent study also showed intersubspecific recombination between coffee-infecting strains in plants intercepted in France . Natural competence could be an explanation for the frequent recombination events detected in X. fastidiosa. Natural competence in X. fastidiosa was recently described in vitro , the rate of homologous recombination was shown to be higher when the cells were growing exponentially in solid agar plates than in batch culture tubes, and minimal medium was more conducive than rich medium . With a plasmid as a donor DNA, 96 bp of flanking homology was sufficient to initiate recombination . Moreover, some competence-related and type IV pili genes were shown to be involved in the process . Although some of those studies were performed using plasmids as donor DNA, two strains were also shown to recombine in coculture conditions , although the capacity of these strains to act either as a donor or a recipient for DNA exchange was not determined in those studies. The objective of this study was to test the hypothesis that natural competence in X. fastidiosa occurs under flow conditions .

Associated with this objective was the aim of elucidating whether previous observations of high frequencies of X. fastidiosa natural competence in vitro were dependent on batch culture conditions , nft growing system which allow cell-tocell contact for longer times without replenishing of nutrients or removal of secreted molecules. Although natural competence and recombination are assumed to occur in natural habitats based on field surveys and DNA sequence data, experimental indications of its occurrence in the plant or insect host are not yet available for X. fastidiosa. Therefore, to circumvent the limitation of X. fastidiosa recombination tests in the natural hosts that are affected by uneven bacterial distribution and low populations , we performed recombination experiments in a microfluidic chamber system that mimics the natural environment of xylem vessels and insect foreguts. The MC system allows continuous media flow conditions and formation of biofilms and has been previously used to study the behavior of X. fastidiosa . The biofilm fraction of the MC and the planktonic and detached cell fraction can be collected separately, and the behavior of cells in the two fractions can be determined. Two strains used in all of the previous publications on this topic were used in this current study to facilitate comparison with the literature and to further our understanding of natural competence in X. fastidiosa. The results presented here show that growth under flow conditions supports natural competence in X. fastidiosa, with recombination frequencies equivalent to that on solid media, previously described to be the most conducive environment for natural competence in vitro . These findings support the hypothesis that recombination occurs at high rates under flow conditions, representing the natural habitats of X. fastidiosa.Bacterial strains, media, and culture conditions. Xylella fastidiosa subspecies fastidiosa mutants NS1-CmR and pglA-KmR were used in this study. The mutants were cultured in periwinkle wilt agar medium , modified by omitting phenol red and adding 1.8 g liter 1 bovine serum albumin  and supplemented with the respective antibiotics. PD3 medium and modified X. fastidiosa medium  were used when stated. Pectin was added to a final concentration of 0.01% as previously described . Kanamycin was used at 30 g ml 1 and chloramphenicol at 10 g ml 1 . Inocula were prepared by streaking cultures from the 80°C freezer stocks on PW agar plates and incubating the plates for 5 to 7 days at 28°C. Cultures were then restreaked onto new plates and incubated for another 5 to 7 days before use. Media selection for natural competence and growth in microfluidic chambers. To select a medium to test the occurrence of natural competence in MCs, three media were first tested in solid agar plates. XFM and PW, used in previous studies , were selected as positive- and negative-control media, respectively, for recombination. Natural competence experiments were performed according to the method of Kung et al. with some modifications. Briefly, cells of the NS1-CmR and pglAKmR mutants were prepared in liquid media by scraping the cultures from PW-antibiotic plates. Ten microliters of each strain was spotted on top of each other on the agar plates of PW, XFM, and PD3 without antibiotics, and the spots were allowed to dry for 1 h. The plates were then incubated at 28°C for 3 days. Next, two spots from the same plate were scraped off and suspended in 1 ml of PD3 to make one replication, and 3 to 4 replicates were included for each media type per experiment. The experiments were repeated independently twice for XFM and at least three times for PD3 and PW. Single mutant strains were included as controls. The suspensions were then serially diluted, and100-l aliquots of appropriate dilutions were plated on PW agar plates in triplicate supplemented with both antibiotics to recover recombinants at the antibiotic-resistant site and with a single antibiotic to check for the growth of both parents in the mixture. Appropriate dilutions also were plated onto PW plates without antibiotics for enumeration of total viable cells. Plates were incubated at 28°C for at least 14 days before CFU were enumerated. The recombination frequency at the antibiotic-resistant site was calculated as the ratio of recombinant CFU to total CFU in equal volumes of suspension. After selection of the media that supported recombination in the agar plates, the media were tested in the MCs for cell attachment and biofilm formation. To test for the specific components that may influence natural competence, an initial screen was performed by removing or adding components to PW and PD3 in solid agar plates as described above. The components tested were sodium citrate dehydrate, succinic acid, and starch ; BSA and L-glutamine ; and pectin. The effect of BSA was further tested by supplementing PD3 and PD3 plus L-glutamine with BSA and removing BSA from PW and XFM. Experiments were repeated three times independently with three replicates each time, except for PD3 plus L-glutamine treatment that was performed once with three replicates. The twitching motilities of both mutants were determined in media with and without BSA, according to previous studies with few modifications.

We used a one-sample t-test to test the null hypothesis that the pollination efficiency of honey bees equals the efficiency of the average non-honey bee floral visitor . Since honey bee relative efficiency did not differ between agricultural and wild plant species , data from all plant species were combined. The best multiple regression model selected from a set of candidate models of environmental variables revealed that the network-level frequency of visits by honey bees is positively related to the first principal component of temperature bioclimatic variables , where higher values correspond with higher overall temperature, higher isothermality, lower annual range and lower seasonality . Honey bees were also more frequent floral visitors in mainland networks compared to island networks . Perhaps surprisingly, our regression model revealed no effect of the honey bee’s native status on honey bee numerical importance . Release from pathogens and parasites often contributes to the success of introduced species ; this factor may be unimportant in honey bees because many of their pathogens have spread worldwide due to trafficking of domestic colonies . Nevertheless, it is noteworthy that eight of the ten networks with the highest relative frequency of honey bee visits come from introduced range localities, and that in five of these networks, honey bees accounted for more than half of the total visits recorded . While Abe et al. found that honey bee dominance in the Ogasarwara satellite islands was driven by an introduced lizard’s preferential predation on native pollinators, black plastic garden pots further studies are needed to understand why honey bees reach high abundance in some parts of their introduced range, but not others.

Also surprising is our finding that study year was unrelated to honey bee numerical importance , given the high mortality in managed honey bee colonies reported over the last two decades . Agents responsible for increased mortality in managed colonies can also affect wild or feral honey bee colonies , but ongoing research also reveals the resilience of unmanaged honey bee populations to mortality agents such as parasites and pathogens . In our pollination networks, the degree to which honey bee individuals are coming from managed versus unmanaged colonies likely varies based on geographical location and proximity of the study site to agriculture. However, in one network with high honey bee numerical importance , genetic testing indicated that the majority of the honey bee foragers were derived from feral, Africanized hives . Although honey bees are numerically dominant pollinators in many networks, their importance as floral visitors to individual plant species varies widely. An examination of 46 pollination networks that provide data on each studied plant species yielded 1629 plant taxa within these networks. While some plant taxa species are found in more than one network, we treat each plant species within each network independently because our goal is to examine the frequency with which honey bees visit each plant species within discrete communities. Across these 1629 plant taxa, we found a strongly, positively skewed distribution of honey bee visitation frequency . Honey bees were the only documented visitors of 5.34% of plant taxa , and contributed the majority of visits to 15.16% of plant taxa . However, honey bees also failed to visit the majority of plant taxa .

Restricting the analysis to plant taxa with ³ 10 visits recorded to minimize extreme values due to low sample size did not qualitatively affect our results . In this data subset, honey bees were the sole documented visitors of 3.44% of plant taxa , contributed the majority of visits to 17.84% of plant taxa , and failed to visit 50.31% of plant taxa . Our finding that honey bees numerically dominated a number of plant taxa is perhaps unsurprising given their ability to recruit nest mates to spatially and temporally abundant floral resources . However, it is noteworthy that this pattern holds true in their introduced range, where floral resources monopolized by honey bees presumably coevolved with native pollinators. This analysis cannot distinguish whether honey bees dominate certain floral resources because they displace other pollinators or because they have the ability to profit from floral resources not valued by other pollinators. However, the data do suggest that honey bees possess the potential to disrupt interactions between plants and other pollinators in the majority of natural communities in which they occur. On the other hand, our finding that honey bees are frequent floral visitors to only a small subset of the plant species in a community is consistent with studies investigating honey bee colony-level resource use and underscores the importance of maintaining robust, diverse communities of non-honey bee pollinators for the persistence of the majority of plant species in natural communities. While our analyses of pollination networks worldwide reveal that honey bees are exceptionally abundant and generalized floral visitors, our analysis of pollination efficiency of honey bees reveals that they are average pollinators with respect to their pollination efficiency . Using a dataset of 35 plant species spanning 23 plant families that exhibit a diversity of flower sizes, shapes, and colors, we compared honey bees and non-honey bee floral visitors with respect to seed set, fruit set, or pollen deposition resulting from single floral visits . The relative pollination efficiency of honey bees did not differ between the 16 agricultural and 19 non-agricultural plant species , perhaps because flowers of agricultural species , squash , tomato often closely resemble those of their wild relatives. Overall, we found no evidence that the pollination efficiency of honey bees consistently differs from that of the average of the non-honey bee floral visitors considered in these studies . Since the importance of a particular pollinator to a given plant species is often calculated as its per-visit efficiency multiplied by its visitation frequency , it seems reasonable, given our results, to assume that the ecological importance of honey bees as pollinators in any community is satisfactorily estimated by their visitation frequency. However, since honey bees are known to exhibit poor efficiency at pollinating certain plant taxa , we caution that careful studies are needed to demonstrate the importance of honey bees as pollinators to particular plant species. Further, in at least one case, high visitation frequency by a pollinator damaged raspberry flowers and led to reduced reproductive success . On plant species and in plant communities where honey bees reach high visitation rates, a similar negative relationship between visitation frequency and plant reproductive fitness may occur and is worthy of investigation . As a numerically dominant, super-generalist pollinator, honey bees may influence the fitness and behavior of competing pollinators, enhance as well as reduce plant fitness, and facilitate the spread of non-native weeds and pathogens . Given the ecological importance of honey bees, there is little doubt that changes in their distribution and abundance will impact the evolutionary trajectory of co-occurring mutualists and competitors, and likely the long-term eco-evolutionary dynamics of communities in which they take part. Our results underscore the urgent need for more data on how honey bees, and the potential loss thereof, shape the ecology and evolution of plant-pollinator interactions on global and local scales.Drosophila suzukii Matsumura is an economic pest of small and stone fruit in major production areas including North America, Asia and Europe. Female D. suzukii oviposit into suitable ripening fruits using a serrated ovipositor. This is unique compared to other drosophilids, square plastic plant pots including the common fruit fly, D. melanogaster, which oviposit into overripe or previously damaged fruit. Developing fruit fly larvae render infested fruit unmarketable for fresh consumption and may reduce processed fruit quality and cause downgrading or rejection at processing facilities. In Western US production areas, D. suzukii damage may cause up to $500 million in annual losses assuming 30% damage levels, and $207 million in Eastern US production regions.

Worldwide, the potential economic impacts of this pest are staggering. Pesticide applications have been the primary control tactic against D. suzukii both in North America and in Europe. The most effective materials are those that target gravid females, including pyrethoids, carbamates, and spinosyns. These applications are timed to prevent oviposition in susceptible ripening host crops. In the Pacific Northwest, many growers have adopted scheduled spray intervals of 4–7 days. This prophylactic use of insecticide is unsustainable as growers have a limited selection of products and modes of action. This could ultimately lead to D. suzukii becoming resistant and may cause secondary pest problems because of negative effects on beneficial organisms. Furthermore, production costs have increased substantially in crops where D. suzukii must be managed. Effective sampling methodology for D. suzukii is lacking despite extensive efforts to improve trap technology or determine effective fruit infestation sampling protocols. Theoretically, traps to capture adult flies should aid growers in the timing of spray applications so that insecticides could be used more judiciously. Traps baited with apple cider vinegar or a combination of sugar-water and yeast are currently used to monitor adult D. suzukii flight patterns. However, without standard methods for trapping or management thresholds based on trap count data, it is questionable how much is gained by establishing and monitoring traps in crops. Establishing, monitoring, and maintaining traps is very labor intensive and the costs do not justify the benefits for many growers. Historically, trap data has not provided a reliable warning against D. suzukii attack, especially for susceptible crops in high-density population areas where considerable oviposition can occur in short time periods. Currently, no significant differences are found in any traps used for monitoring D. suzukii given differences between crops and environments where traps have been tested. Monitoring fruit infestation levels to guide management may also be impractical. It is unclear how many samples would be needed to accurately determine infestation levels. Furthermore, by the time larvae are detected in the fruit, it is too late for management action and damage has already occurred. No detailed studies could be found using monitoring for fruit infestation for this pest, and precision of sampling methodology is currently unavailable. Degree-day , or phenology models, are standard tools for integrated pest management in temperate regions and are used to predict the life stages of pests in order to time management activities and increase the effectiveness of control measures. Degree-day models work best for pests with a high level of synchronicity and few generations. Our data suggest that D. suzukii has short generation times, high reproductive levels, and high generational overlap compared to other dipteran fruit pests. Given this life history, stage-specific population models represent an alternative and potentially more applicable tool for modeling pest pressure. Pest population estimates may be greatly improved by employing additional tools such as mark recapture and analytical or individual-based models. The ability to describe and forecast damaging pest populations is highly advantageous for fruit producers, policy makers, and stakeholder groups. Many such studies have been directed at forecasting populations of medically important insect species. The major factors affecting survival, fecundity and population dynamics of drosophilids include temperature, humidity, and the availability of essential food resources. Therefore, an improved understanding of the role of temperature on D. suzukii may provide for a better understanding its seasonal population dynamics. In this paper, we present a population model for D. suzukii that represents a novel modification of the classic Leslie projection matrix, which has proven to be one of the most useful age structured population models in ecology, with applications for diverse organisms including plants, animals, and diseases. Our modification accounts for the effect of temperature on the survival and fertility of D. suzukii in calculating population growth of the organism. Typically, researchers have introduced elements of environmental stochasticity to matrix models to study environmental effects on population trajectories. However, our approach relies on temperature-dependent estimations of age specific fecundities and survival that are determined by models fit to life table data generated for multiple temperatures. Our environmentally dependent matrix model is unique in that it does not rely on simulation of environmental effects on populations, but the matrix itself is recalculated at each iteration in direct response to environmental input. Model predictions were run under environmental conditions from different regions to illustrate variation between and within study sites in different years. These simulations make important predictions about age structure and population trends that have implications for pest management both in a broad sense and with regional specificity.

Accounting for abundance makes multivariate dispersion less sensitive to rare species, which often make up a large fraction of the total species richness in bee assemblages but may contribute little to the pollination services rendered to plants . For these reasons, multivariate dispersion is superior to the traditional approach of using multiplicative or additive partitioning for investigating bee temporal beta diversity with respect to characterizing individual-level bee assemblage composition, as well as temporal turnovers in ecosystem function. To calculate multivariate dispersion, we performed a non-metric multidimensional scaling ordination based on a dissimilarity matrix of abundance-weighted bee assemblages in all possible pairs of samples across all plots . From this ordination, we calculated the multidimensional centroid of the samples from each plot, and then computed the mean distance between each plot’s centroid and its constituent samples. The resulting dispersion score for each plot thus measures the degree to which the species composition of each plot’s bee assemblage turns over through time. Dispersion scores of reserve and fragment plots were then compared using Welch’s two sample t-tests. As with our analyses of temporal gamma and alpha diversity, we repeated all analyses with the temporal beta diversity of native plants as an added independent variable . Models that included plant temporal beta diversity yielded poorer AICc scores in all cases; thus, we did not include plant temporal beta diversity in our final models.Across our two years of sampling, we found consistent differences in bee assemblages occurring in reserves and fragments, despite the known tendency for bee faunas to exhibit considerable inter-annual variation at a given locality . Compared to reserves, big plastic pots fragments harbored bee assemblages that were less diverse with respect to all three components of temporal diversity .

While all metrics of bee diversity and abundance varied with time, differences in bee diversity between reserves and fragments were remarkably constant through time . Individually scrutinizing the three components of temporal diversity allowed for a high resolution characterization of the temporal structure of bee assemblages in intact and fragmented habitats; these analyses also yielded further insights into the potential consequences of bee diversity loss for ecosystem function in fragmented habitats in our system. Reduced species richness is one of the most commonly reported effects of habitat fragmentation on bee assemblages . Though our reserve and fragment plots did not differ systematically with respect to the composition of floral resources , it is possible that decreased availability of nest sites within foraging distance of key host plants or increased vulnerability to demographic stochasticity due to isolation or small population size may have contributed to reduced bee species richness in fragments. Analyses of the temporal gamma and temporal alpha components of bee species richness yielded qualitatively similar results; however, the impact on each of the two temporal diversity components may have distinct implications for the conservation of bees andecosystem function. The temporal gamma component of bee richness provides information on the habitat conditions and locations that support the greatest total number of bee species or species of particular conservation concern; as such, it is the most useful metric for developing conservation strategies aimed at bees. On the other hand, the pollination effectiveness of a particular bee species for a particular plant species may depend upon the timing during which the interaction between bees and plants takes place or upon the bee species’ functional complementarity with other, temporally co-occurring pollinator species .

Detecting potential impacts of climate change on the phenological matching between bee species and the plants they pollinate also requires examining the composition of bee assemblages at discrete points in time. Thus, in the face of a changing climate, effective strategies aimed at conserving bees and the ecosystem function they perform should account for both the temporal alpha and gamma components of bee richness. As with patterns of bee species richness, patterns in the temporal gamma and alpha components of bee assemblage evenness are in qualitative agreement with each other. Assemblage evenness is an important driver of ecosystem function , including pollination , but remains an under-appreciated aspect of pollinator assemblage dynamics . Reductions in the temporal alpha component of bee assemblage evenness in fragments may result in decreased frequencies of interspecific encounters among bee species; such encounters have been shown to enhance pollination efficiency via altering bee foraging behavior . On the other hand, reductions in the temporal gamma component of bee assemblage evenness may result in a stronger reliance by plant assemblages on a small subset of numerically dominant bee species, and consequently, reduced stability of pollination services . In contrast to patterns of bee species richness and assemblage evenness, overall bee abundance did not differ between reserves and fragments. This pattern was caused by reserves having higher bee abundance in spring and fragments having higher bee abundance in summer . This treatment-by-sample interaction appears to be driven by the higher relative abundance of generalist bees in fragments ; many generalist species in our system reach peak abundance between late June and August. Generalist bees may be more tolerant of habitat fragmentation compared to specialists and have been hypothesized to replace the ecosystem function formerly performed by extirpated specialists .

However, even though generalists in our study numerically compensated for absent specialists when considering the temporal gamma component of bee abundance , reduced bee abundance in fragments early in our study period may threaten the pollination of spring blooming plant species. Temporal beta diversity represents another under-appreciated metric in ecology , and reports on the effects of anthropogenic disturbance on intra annual turnover of biological assemblages remain rare . In our system, decreased temporal beta diversity in fragments may explain how modest reductions in the temporal alpha component of species richness and assemblage evenness in fragments translate into more pronounced reductions in the temporal gamma component . More broadly, decreasing seasonal turnover in an assemblage may result in increasing temporal niche overlap among its constituent species , which may in turn decrease the number of distinct temporal niches created by the assemblage. Decreases in the seasonal turnover of bee assemblages may be especially consequential in cases where bee species tend to interact with a set of preferred host plants throughout their activity season even when new plant species begin to bloom as time progresses . If temporal host-switching is likewise rare in our system, reduced bee assemblage turnover in fragments may jeopardize the reproduction of certain plant species that occupy specific temporal niches with respect to pollination . Examining the temporal beta diversity of bee assemblages thus appears crucial for understanding mechanisms underlying the impact of anthropogenic disturbance on pollination services. Novel selection forces in altered habitats often create ecological filters —environmental or biotic processes that determine which species can establish or persist. The strength of ecological filters depends on the form of disturbance, the natural history of organisms in question, and the strength of other forces shaping community assembly such as dispersal, competition, and natural disturbance regimes . Understanding the extent to which ecological filters shape community assembly represents a central goal of community ecology , and can be used to predict the long-term implications of habitat modifications . Assessing the strength of ecological filters is especially important when evaluating the long-term ecological consequences of habitat fragmentation, one of the leading causes of ecosystem change and biodiversity loss worldwide . Many studies have found strong evidence that ecological filtering drives diversity loss in habitat fragments, both at the local scale and landscape scale . On the other hand, community assembly in fragmented landscapes may also be shaped by stochastic colonization and extinction events typical of island biogeography , or by underlying heterogeneity among habitat patches . While habitat fragmentation could reduce local species richness through different combinations of the above mentioned processes, effective conservation practices will need to account for the degree to which ecological filtering drives species loss . One powerful approach to assess the strength of ecological filters in fragmented habitats is to examine functional diversity, since ecological filters, by definition, black plant pots plastic act on functional traits rather than species . Functional diversity is related to taxonomic diversity in complex ways , and the relationship between the two metrics can provide insight into the mechanisms that drive biodiversity loss in fragmented habitats. For instance, when habitat fragmentation results in strong ecological filtering, functional diversity may decline even if species richness and abundance remains little altered, as may be the case when taxa that thrive in fragmented habitats replace those that are extirpated .

On the other hand, if species loss in habitat fragments mainly results from stochastic extinction events associated with small population size and isolation, functional diversity may be relatively unaffected by the loss of taxonomic diversity, especially in systems with sufficient functional redundancy among species .Here, we evaluate the contribution of ecological filters to diversity loss by taking advantage of an extensive survey of native bees in a species rich ecosystem where we have documented profound reductions in bee species richness associated with urbanization-induced habitat fragmentation . Bees are ecologically important pollinators known to exhibit non-random species loss in fragmented habitats, where specialist species appear particularly vulnerable . Ecological filtering of bees may occur in habitat fragments when fragments experience reductions in the diversity or abundance of plant species that serve as food resource for bees, exhibit altered abiotic conditions due to influences from the surrounding matrix , or fail to contain the correct spatiotemporal configuration of food and nesting resources . Habitat fragmentation may also reduce bee diversity via processes not related to ecological filtering; for instance, when the isolation of habitats disrupts dispersal processes crucial in buffering bee populations from year-to-year variation in the local and temporal distribution of floral resources . We assess the strength of ecological filters by addressing four questions. First, to what extent does fragmentation impact bee functional diversity? Loss of functional diversity more severe than that predicted by a null model of random species loss would lend support for the importance of ecological filters . Second, do bee assemblages in fragments exhibit distinct taxonomic or functional compositions compared to those in reserves, as would be expected when ecological filtering causes the assembly of novel communities ? Third, do bee assemblages in fragments exhibit lower taxonomic or functional beta diversity among plots, as would be expected when ecological filters select for or remove common sets of functional traits in altered habitats ? Lastly, are bee assemblages in fragments composed of taxa with larger range sizes relative to those in reserves? Given that range size tends to be positively related to niche breadth , a shift to more cosmopolitan species in fragments is expected if ecological filtering precludes the persistence or colonization of species more specialized to the local ecosystem . Answering these questions will yield insights into the mechanisms that drive bee species loss in our study system as well as provide information on the potential conservation value of scrub habitat fragments .Study system: Field data were collected between April and August of 2011 and 2012 in coastal sage scrub habitat in San Diego County, California, USA, and are detailed in Chapter 1 of this dissertation. We surveyed one-hectare study plots belonging to two categories: large natural reserves , and habitat fragments surrounded by urban development. This is the same system of reserves and fragments previously used to study the ecological effects of urbanization-induced habitat fragmentation . In 2011, we surveyed eight study plots ca. every 2-4 weeks; in 2012, we surveyed 17 study plots ca. every 3-5 weeks. During each survey at each study plot, the first author deployed 30 bowl traps between ca. 0900 h and 1500 h, and collected free-flying wild bees via aerial netting . Concurrently, we also documented the identities of native, insect-pollinated plant species in bloom at each study plot by walking through pre-planned paths that allowed an observer to visually survey the entire study plot . All collected bees were individually mounted and identified to species or morphospecies within genus, hereafter referred to collectively as “species” . This sampling effort resulted in a dataset of 11,037 native bees belonging to 216 species in 52 genera and 6 families, after the exclusion of bee specimens not identifiable beyond genus .