Horticulture

The transfer and extension of ideas and concepts in order to bring new topics under the compass of existing theory has obvious scientific merit . It also has pitfalls. The failings of early “evolutionist” models of social evolution and their archaeological adaptations, as well as social Darwinist interpretations, are well-rehearsed subjects in anthropology. Contemporary anxieties about the use of neo-Darwinian theory in anthropology are more narrowly and analytically focused, and sometimes not so easy to set aside. A recent example would be debate over the claim by Rindos that his co-evolutionary account of plant domestication had successfully banished human intent from an explanatory role in this process . In the present volume we take for granted the relevance to agricultural origins of neoDarwinian and behavioral ecology theory. We reject without explicit argument the sub-stantivist claim of economic anthropology that none of the tools of formalist, microeconomics has any purchase outside of modern capitalist economies . To the contrary, we believe it evident that the basic concepts of HBE are fundamental to the analysis of any economy. Close attention to their use in HBE we believe will stimulate new applications and models specifically designed to analyze mixed economies and food production. We are more receptive to the argument that specific foraging models, developed as they were for foragers, may be only partially appropriate to the analysis of emergent food producers. For instance, raspberry plant container the diet breadth model assumes random encounter with resources, a condition increasingly likely to be violated as foragers become involved in the manipulation of individual species.

In as much as all models simplify reality and thus violate at least some conditions of their application, the unavoidable judgment is this: does the failure to fit this particular assumption completely vitiate the heuristic or analytical value of the model? With the specific cautions cited in individual papers, we believe the combined weight of the case studies developed in this volume add up to a strong presumption in favor of the utility of foraging theory, even as the foragers being analyzed direct more and more of their effort toward agricultural activities. We envision three levels where HBE might be applied to the question of agricultural origins. Extant models, although designed for the analysis of foraging, might be applied in the analysis of agricultural origins with little or no alteration in their structure and assumptions. This is the procedure of most authors in this volume. Extant models might be modified so to more directly address questions or situations specific to non-foraging aspects of economy, including cultivation and agricultural production. The modification of central place foraging models to analyze the question of field processing is an especially good example of this. Finally, entirely new models, inspired directly by the problem of explaining human subsistence transitions, might be devised using fundamental behavioral ecology concepts such as opportunity cost or discounting. We think of these options as adopt, adapt, or invent, respectively.

While options and hold great potential for novel and perhaps quite interesting analyses, it appears from the papers assembled here that there is much to be accomplished with the simple adoption of existing models.The fresh market berry industry in Santa Cruz and Monterey counties is an excellent example of transformation in the business of agriculture over the last 50 years. Located along the Central Coast of California, the two counties span the fertile Pajaro and Salinas valleys, and are well known for their amenable climate and production conditions, their diverse crop mix and grower demographics, and their developed agricultural infrastructure and support industries. The majority of the berry sector is comprised of strawberries , raspberries and blackberries , with blueberries and other miscellaneous berries produced on a much more limited basis. Substantial research-based literature and historical information is available for Central Coast strawberries; however, despite the area’s move towards greater production of raspberries and blackberries, less information exists for these crops. We seek here to provide a more complete portrayal and historical context for the berry industry in the Santa Cruz and Monterey area, which is the origin of the berry industry in California. While the berry industry has been very successful in recent decades, it now faces new challenges, such as invasive pests and the phaseout of the soil fumigant methyl bromide. This article draws on previous and more recent research to discuss some of the influences that have contributed to the berry industry’s dramatic expansion in Santa Cruz and Monterey counties, including selected innovations in agricultural practices and heightened consumer demand. During the 1960s and 1970s, the number of acres planted to berries, tons produced and value of production fluctuated. The fluctuations can be partly explained by farm management: in the past growers often rotated berry and vegetable crops to assist with soil and pest management, thereby influencing these statistics. However, annual crop reports from the county agricultural commissioners show that since the 1980s, berries have become increasingly important to each county’s overall value of production, and by 2014 accounted for 64% and 17% of the total value of all agricultural products in Santa Cruz and Monterey counties, respectively .

The industry’s growth can be explained by a shift of some acreage out of tree fruits and field crops , among others, into berries, and by additional acreage put into agricultural production. Strawberries are the undisputed leader in the berry sector and in 2014 represented 58% and 94% of the value of all berry production in Santa Cruz and Monterey counties, respectively , and 50% and 93% of all berry acreage . Table 2 documents the remarkable expansion of the strawberry industry over time in both counties with respect to acreage, tons produced and value of production. Between 1960 and 2014, acreage more than tripled and production increased tenfold. The value of production, in real dollars, increased by 424% in Monterey County and by 593% in Santa Cruz County, reaching an astonishing combined value of nearly $1 billion in both 2010 and 2014. The gains in all statistical categories in Monterey County were enabled in part by an expansion of production into the southern reaches of the county where more and larger blocks of farmland are available, container raspberries and where land rents are lower than in Santa Cruz and northern Monterey counties. However, from 2010 to 2014 Monterey County’s tonnage and production values declined, possibly because the area has recently experienced a shortage of labor to harvest fresh market crops. Tonnage was also lower in Santa Cruz County, but production values increased. This may be because of the county’s greater emphasis on local agriculture, organic production and direct market sales, which are often associated with higher crop values. For raspberries, the acreage, tons produced and value of production grew steadily and most strikingly in Santa Cruz County , where production conditions for caneberries are optimal. For example, caneberry fields in Santa Cruz County are situated in areas that have well-drained soils and are protected from damaging winds. Also, fields are planted to take advantage of the growth and yield gains associated with southern exposures. Moreover, field-to-cooler travel distances are shorter in Santa Cruz County, which is critical for safeguarding the quality and marketability of these highly perishable crops. By 2014, raspberries represented 33% of the county’s total value of production for all berries. In contrast, Monterey County raspberry production accounted for only 6% of the county’s total berry value. Blackberries have not been consistently reported as a separate category in archived statistical analyses, but instead were often included under the terms “bush- or miscellaneous berries”. Therefore, similar data for blackberry acreage and value of production cannot be reported here. However, between 1990 and 2010, Santa Cruz County agricultural commissioner crop reports reported an upward trend for the broad category with respect to acreage planted and value of production . In 2010, blackberries were promoted to a position of prominence in the report and shown as a separate statistic; at the same time, the miscellaneous berry category was shown to be very small indeed. Between 2010 and 2014, however, blackberry acreage and value of production leveled off and have shown only modest gains . This may be because there has been less emphasis on production and market research and promotion for blackberries than for strawberries or raspberries. No comparable data are available for Monterey County. The two counties have contributed significantly to California’s total berry sector: in 2014, area strawberry acreage represented 35% of the statewide total, 37% of the total tons produced and 38% of the total value of production . Area raspberry acreage represented 43% of the statewide total, 42% of the total tons produced and 39% of the total value of production. Comparable statewide statistics are not available for blackberries. County agricultural commissioners’ reports show that the majority of all berries produced in the two counties — up to 98% — are sold as fresh market fruit . In years with adverse production conditions or low prices, a higher percentage of the crop may be diverted to the freezer or processed products market. Fresh market fruit is handled and sold primarily through local grower-shippers; a much smaller share is sold directly to consumers through farmers markets, community supported agriculture operations, farm stands and other direct and intermediated market channels such as restaurants, independent grocers and schools.

Arguably the most momentous shift in cultural practices for strawberries was the introduction of preplant soil fumigants, beginning with chloropicrin in the 1950s and methyl bromide in the 1960s. Fumigation is a soil disinfestation practice that improves plant productivity and helps with the management of arthropods, nematodes, weeds, soilborne fungi and other plant pathogens. Some of the most difficult to control pathogens include Verticillium dahliae, Fusarium spp. and Macrophomina phaesolina. Without soil fumigation, these pathogens have the potential to completely destroy strawberry plantings. Early on, when CP and MB were mixed and applied together, the synergistic effects allowed strawberries to be produced as an annual rather than a biennial crop, and to be grown continuously on the same land without rotation to another crop, resulting in an increase in annual strawberry acreage. The use of fumigants also led to higher and more predictable yields and fruit quality, and further enabled the development of more stable markets for strawberries . Yields for strawberries statewide increased from a range of 2 to 4 tons per acre prior to the introduction of soil fumigants to 16 tons per acre by 1969 . Additional cultural improvements included the development of both UC and proprietary strawberry varieties uniquely adapted to coastal production conditions. Varieties were bred, for example, for disease resistance, yield and market potential. Notable UC-bred strawberry varieties include Tufts , Pajaro, Douglas, Chandler, and Selva , Camarosa and Seascape , and Aromas, Albion and Monterey . Irrigation practices also evolved, shifting from furrow irrigation in the 1960s to drip irrigation in the 1980s, which led to further improvements in plant disease management and greater water use efficiency. These and other enhancements meant that by 2012, yields could exceed 35 tons per acre . More recently, the strawberry industry has focused on “fine-tuning” fertility and water management for more efficient resource use, along with additional yield and fruit quality improvements . The Santa Cruz–Monterey area is also recognized for its early experience with conversion of conventional strawberry production to organic management . Organic strawberry production was shown to result in lower yields, which, when offset by premium prices could potentially offer higher net returns to growers. The importance of crop rotation for disease management was not addressed in the initial study by Gliessman et al. but has since been the focus of additional research, as have more complete analyses of the economics of organic strawberry production . Growers and area researchers continue to collaborate and advance organic strawberry production techniques. Most notably, a long-term research commitment has been made to determine organically acceptable disease management practices such as anaerobic soil disinfestation , the use of commercially available soil-applied biological organisms and the incorporation of soil amendments such as mustard seed and its derivatives. The area is now seen as a global leader in organic strawberry research, and in 2012 the first organic strawberry production manual was published by UC Agriculture and Natural Resources . Statistics documenting expansion of the organic strawberry industry over time are not available on a county-by-county basis, but statistics for California show prodigious growth in acreage and value of production: from $9.7 million in 2000 to $93.6 million in 2012, a 621% increase in real dollars .

Similarly, a comparison of herbivory damage on herbarium specimens of non-native plants in urban versus rural environments might provide insights into one pathway towards species invasion. Many non-native plant species are introduced into urban areas, and urbanization may provide warm, enemy-free space where they can establish and subsequently expand. Though the role of urbanization in natural enemy release and subsequent invasions is not well characterized, we might predict that non-native plants escape their natural enemies in urban areas and experience increased herbivory rates when they move into natural areas where they encounter a higher diversity of herbivores.Responses to global change, such as those in space and time, discussed above, encompass plasticity in behaviour or physiology and distributional shifts, which may be rapid. However, there is growing evidence that evolutionary responses might also be rapid, assuming there is sufficient standing genetic variation for selection to act upon. While fluctuating selection can maintain this standing variation, providing the raw material for future adaptive responses, strong directional selection, such as that imposed by anthropogenic climate warming, plastic gardening pots can erode genetic variation and potentially impede evolutionary adaptation, elevating population extinction risk.

Insect herbivores and the plants they feed upon are locked in an evolutionary arms race, and insect herbivory drives contemporary plant evolution, changing plant allele frequencies within a few generations. It is likely that climate-induced shifts in herbivory will impose additional selection on both plants and insect herbivores already under pressure from direct effects of climate change. Natural history collections that span multiple generations can provide a record of evolutionary changes and constraints. It can be difficult, however, to disentangle plastic and evolutionary responses. Evolutionary responses can be predicted from the breeder’s equation, but this requires extensive long-term population data. For species that can be stored in a dormant state, such as plants, it is possible to contrast ancestral and descendent genotypes grown under common conditions, and Franks et al. were able to demonstrate evolution to earlier flowering in Brassica using stored seeds. Seedbanks and other collections that hold propagules, intentionally or incidentally, could thus provide important data for exploring evolutionary responses and testing whether species might be approaching limits in their adaptive responses. Sequencing of archived tissue of plants and animals already allows for the signature of selection to be sought directly in their DNA. New collections could systematically sample seeds through time or across populations, providing the potential to resurrect past populations and examine micro-evolutionary change.

Evolutionary insights from herbarium specimens might be particularly useful for adapting agricultural practices with global change. Alongside the insights that collections data can provide on ecological and impacts of global change in natural systems, herbaria are additionally repositories of crop wild relatives . CWR are important sources of phenotypic and genetic information on pest and disease resistance that may be introgressed into crops. For example, comparative analyses of CWR might provide an opportunity to identify herbivore-resistance traits relevant to agricultural and ornamental species, such as glandular trichomes that act as physical defences against insects and can be detected on herbarium specimens with a microscope. Herbaria provide a record of this genetic diversity even when it is no longer present in the wild. In addition, herbivore damage on CWR herbarium specimens might help predict increases in pest pressure on crops, because closely related host species tend to be vulnerable to similar suites of pests and pathogens. Specimens in herbaria can also serve as records of past biotic threats and inform how we can avoid these threats in the future. For example, Yoshida et al. sequenced the genomes of Phytophthora infestans, the cause of potato late blight, infamous for its role in the Irish Potato Famine, from herbarium collections of infected potatoes and tomatoes.

Using genomic tools, they found one strain of P. infestans linked to the potato blight in the nineteenth century, but that multiple strains moved globally in the twentieth century. In a recent study, we quantified historical insect damage on a crop species, the low bush blueberry, Vaccinium angustifolium, growing in the wild to determine how pest pressure has changed with recent climate change. The low bush blueberry is an ecologically and economically important endemic species in northeastern North America, whose production has seen recent increases owing to awareness of the health benefits of blueberries. Collection records from the Harvard University Herbaria suggest that herbivore damage has increased in recent years, with evidence that increased herbivory is a result of winter climate warming. This highlights the need for increased monitoring of herbivore species on V. angustifolium and allows the development of proactive pest management practices that could be implemented before economic impacts are felt.Given the millions of plants and insect specimens that are becoming available online, it will increasingly be possible to assess changes in phenological synchrony, distributions, and occurrence over time across diverse taxa and large spatial areas. The sampling of species within museums and herbaria, however, is non-random and often sparse, which can present distinct challenges depending on the response variable of interest and how robust the data are for answering particular questions. However, the depth of sampling within natural history collections is difficult to assess because natural history collections data are often dark—without searchable databases—despite efforts to rapidly digitize. Another obstacle is that data associated with museum specimens can have large uncertainty; for example, specimens collected before the advent of geographical information system technology often have only coarse scale location data that may prohibit local-scale analyses. When assessing phenological change, the most important challenges arise because of biases in collecting. Herbarium specimens are more likely to be collected near roads; rare species are, perhaps unsurprisingly, collected less frequently, and collections are more likely to be made in spring or summer months. Such biases can make specimen data difficult to work with. For example, roads might be warmer than the surrounding countryside, and observations of shifts in phenology through time might, in part, also reflect the increasing extent of the road network. Finally, sampling frequency may bias estimates of first events, because we are more likely to observe earlier events with greater sampling intensity. Such sampling biases can make it difficult to compare across species when sampling effort varies, for example, between common and rare species. New methods offer a solution to such challenges. For example, methods have been established for calibrating species distribution models according to known biases in presence-only data, and newly constructed statistical models allow robust estimates of the tail of a distribution—in the context of phenology, first flower, for example—even when sampling is uneven. Shifting collection practices may also introduce biases. Herbivory measurements derived from herbarium specimens are probably underestimates in most cases because collectors try to avoid collecting damaged specimens. Even so, herbivory is prevalent on specimens and matches patterns derived from theory and observations from living plants. Importantly, biases introduced by collectors are not necessarily problematic if they do not vary across axes of interest. For example, if collectors are equally likely to collect relatively undamaged specimens across latitude, blueberry pot size herbarium specimens might still provide insights into how herbivory varies with latitude. When there are concerns that collecting practices may have influenced observations—for example, perhaps collectors are more or less likely to collect damaged specimens over time—collector identity may be added to statistical models to partially control for such biases. A unique challenge to using herbarium specimens is that they are eaten by a suite of insects within museums. This loss of material can reduce the use of insect specimens for morphological and genetic analyses. For plants, chewing herbivory created indoors, after a plant was collected, can be confused with damage created while plants were alive. We developed protocols that allowed us to reliably discriminate chewing damage created pre- and post- collection described in.

Such approaches, however, require careful examination of specimens with a microscope and entomological knowledge to recognize diagnostic features of damage to plants, which is a barrier to large scale, rapid scoring of chewing damage on digitized specimens. Other types of insect damage, such as leaf mines, skeletonization and galls, are almost never a product of insects eating plants within museums, and might be scored more reliably, although their prevalence is lower.We have previously argued that herbaria should be repositioned as hubs for ecological research, and we provided suggestions for how to manage collections to promote ecological research on global change. Because the vast majority of research in collections has historically been on taxonomy and systematics, collections are rarely faced with providing data for ecologists and evolutionary biologists. Stronger relationships between researchers in the field, collections managers, and digital data providers would help ecologists to better address the challenging ecological questions of global change, and, importantly, could also increase funding opportunities for maintaining and building natural history collections, which are often under-funded and threatened by institutional priorities. Here, we provide three suggestions for how natural history collections and ecologists can work together to support global change research on species interactions. First, it would be helpful to detail the sampling protocol used to collect specimens. In many cases, collecting practices are haphazard, but if, for example, a curator collects a specimen expressly to document a gall, this would change the inferences we can make from this specimen . Second, ecologists could engage with curators in projects that involve resampling areas and taxa that have long historical records, ideally at the same time of year and with the same research effort involved in previous collections. Third, specimens are most useful when researchers can associate collections with important predictor variables representing species traits or abiotic data related to global change. New opportunities exist to link specimens to the published literature and trait databases , BIEN as well as non-traditional data sources, such as written records and historical photographs. The better integration of new bio-informatics tools and digital databases within biological collections will help transition museums and herbaria into ecological data centres.The volume before you is the first systematic, comparative attempt to use the concepts and models of behavioral ecology to address the evolutionary transition from societies relying predominantly on hunting and gathering to those dependent on food production through plant cultivation, animal husbandry, and the use of domesticated species embedded in systems of agriculture. Human behavioral ecology is not new to prehistoric analysis; there is a two-decade tradition of applying models and concepts from HBE to research on prehistoric hunter-gatherer societies . Behavioral ecology models also have been applied in the study of adaptation among agricultural and pastoral populations. We review below a small literature on the use of these models to think generally about the transition from foraging to farming, while the papers collected here expand on these efforts by taking up the theory in the context of ethnographic or archaeological case studies from eleven sites around the globe.There are older transformations of comparable magnitude in hominid history; bipedalism, encephalization, early stone tool manufacture, and the origins of language come to mind . The evolution of food production is on a par with these, and somewhat more accessible because it occurred in near prehistory, the last eight thousand to thirteen thousand years; agriculture also is inescapable for its immense impact on the human and non-human worlds . Most problems of population and environmental degradation are rooted in agricultural origins. The future of humankind depends on making the agricultural “revolution” sustainable by preserving cultigen diversity and mitigating the environmental impacts of farming. Simple population densities tell much of the story. Huntergatherers live at roughly 0.1/km2 ; rice agriculturists in Java at 1,000/km2 , a ten-thousand-fold difference. There were an estimated ten million humans in the world on the eve of food production ; now oversix billion people live on this planet, an increase of 600% in only ten millennia. Agriculture is the precursor, arguably the necessary precursor, for the development of widespread social stratification, state-level societies, market economies, and industrial production . Social theory maintains that present-day notions of property, equality and inequality, human relationships to nature, etc., are shaped, at least in part, by the social organization, technology, or food surpluses entailed in our dependence on agriculture.

Random effects were modeled with plant identity nested within site to account for the block design of the experiment and to control for variation between our sites. To model count data and to correct for over dispersion, we used a Poisson-lognormal model with a log link function by including a per-observation random effect as described above .We modeled CBB removal by ants using a GLMM. We included treatment , coffee plant distance to nest tree, ant activity on coffee plants after string placement, the interaction between treatment and distance, and the interaction between treatment and ant activity on plants as fixed effects . Random effects were modeled with plant identity nested within site to account for the block design of the experiment and to control for variation between our sites. To model count data in our response variable we used a Poisson distribution with a log link function.We constrained model selection to include biologically pertinent terms for inference and to aid in model interpretation. A full model of these terms was tested, along with subsequent models of different covariate combinations and a null intercept-only model of random effects . The best fit model was determined via backwards model selection compared to the full model, blackberries in containers where the model that resulted in the lowest AIC score with ∆AIC > +2 was selected. Overall significance in models was assessed using Wald type II Chisquared tests. Statistical differences among treatments were compared by Wald Z tests .

In all cases, fixed effect parameters and the variance of random effects was estimated by maximum likelihood with Laplace approximation using the ‘glmer’ function in the ‘lme4’ package in R . To aid in data interpretation, we removed one coffee plant replicate from our analysis where measured ant activity was more than double that of any other plant measured and may have resulted from an unusually high buildup of scale insects which are tended by A. sericeasur on coffee. Additionally, one nest tree replicate was not included in the tree activity analysis because the data were not collected at that site. Finally, coffee plant height and distance to nest tree were centered and scaled to aid model interpretation. All analyses were performed in R .Our experiment demonstrates that the addition of string to connect shade trees and coffee plants in coffee agroecosystems facilitates movement for A. sericeasur and potentially increases ant recruitment rates. Studies in natural systems have reported increases in ant activity with arboreal connections across the arboreal stratum , possibly driven by the easy access these pathways provide to resources . Other ants, such as Pogonomyrmex spp. prefer linear arboreal substrates and switch to cleared routes as a mechanism to reduce the energetic costs of ant foraging , and in some cases to decrease the risk of encountering predators . The observed increase in ant activity on connected coffee plants after the placement of strings suggests that structural connectivity can increase ant recruitment rates to foraging areas in coffee and may enhance the efficiency of movement for A. sericeasur.

This may lead to increased foraging efficiency for ants and enhanced resource capture rates on coffee. However, this could also reflect other benefits associated with using linear arboreal substrates, such as avoiding predators, a behavior that is known to occur in A. sericeasur . Using more efficient foraging pathways and thereby avoiding the leaf litter as a primary foraging substrate may potentially protect A. sericeasur workers from the attack of the phorid fly parasitoid Pseudacteon spp. . While ant activity only significantly increased after string placement on connected coffee plants, we also observed lesser increases in ant activity on control coffee plants and nest trees . This unexpected result could mean that strings, a novel element in the environment, acted as a form of habitat modification or disturbance, which increased overall ant activity in the local area. However, if our manipulation were the cause, we would have expected the ants to attack the jute strings , a behavior that we did not observe during the experiment. Experiments in tropical forests have shown that the long-term removal of lianas can influence ant richness on trees , and therefore may possibly also affect overall ant abundance and activity when promoted. It is also possible that other factors could potentially explain this result in control plants, such as changes in local abiotic factors that we did not measure systematically in our experiment. Future research which expands on the temporal scope of this study may be useful in assessing the long-term effects of artificial connectivity in this system. Ant activity post string placement was negatively affected by distance to the nesting tree .

This result is consistent with previous studies suggesting that within 5 meters A. sericeasur dominance in the leaf litter decreases with distance to the nesting tree . However, in our study, the effect of distance after string placement was significant only on control plants, but not on connected plants. This suggests that connections could buffer the negative effects that larger distances from the nesting tree pose to ant activity and potentially increase ant-provided biological control services in these plants. Connected coffee plants also had significantly higher CBB removal than control plants . Overall, greater ant activity on coffee plants was associated with higher CBB removal rates , suggesting that ant activity directly influenced CBB removal rates. However, while this effect was significant on control coffee plants it was only marginally significant on connected plants. While we believe that these results support the hypothesis that connectivity enhances ant foraging and bio-control services on coffee, the use of dead CBB in this experiment as a proxy to measure bio-control may explain the only marginally significant effect of ant activity on CBB removal in connected plants. It is possible that dead prey exhibit more variable recruitment responses from ants than live prey. Despite this, it is likely that strings facilitated ant movement to coffee plants by providing a smooth, linear substrate and indirectly increased CBB removal . In other systems, the leaf-cutting ant Atta cephalotes uses fallen branches to rapidly move between areas and thereby quickly discover new food resources . Similarly, these resources allow scouts to return quickly to the colony, minimizing the time taken for information transfer and recruitment of other foraging workers . The role of trunk trails and fallen branches has received extensive attention in the leaf-cutting ant system, however, fewer studies have looked at the influence of connectivity resources on foraging behavior of predatory arboreal ants. Surprisingly, CBB removal did not follow the same trend as ant activity with distance to the nesting tree. While control plants tended to have lower CBB removal rates than connected plants as distance to the tree increased, blackberry containers we did not find a significant effect of distance on CBB removal in either control or connected plant groups. Collectively, these results suggest that connections in the arboreal stratum have the potential to increase ant activity and therefore enhance plant protection from CBB attack, particularly in connected plants. Further studies should assess the effect of distance on CBB removal using plants located at distances larger than 3.5 m from the tree. It is important to note the potential for negative impacts of enhanced ant activity on coffee plants resulting from hemipteran tending behavior. While ant activity can increase the density of green coffee scale insects, which may harm coffee plants and reduce coffee productivity, the scale are not a major pest in the region of study, and are not as economically significant as the coffee berry borer . A recent study evaluating the benefits associated with the indirect Azteca–Coffea mutualism, which emerges from the Azteca-scale mutualism, found that the protective benefit ants provide to coffee plants is positively associated with high densities of the scale . Therefore, it is possible that enhanced CBB control associated with scale-tending by ants outweighs the costs associated with scale damage. However, these interactions may be context-dependent, and still need to be fully evaluated in the field to provide a holistic understanding of the impact of connectivity on scale density and coffee yield. Other ant species could also benefit from the addition of connections between coffee plants and shade trees, such as Cephalotes basalis and Pseudomyrmex simplex, which were observed using these connections during our study.

The ant P. simplex has been previously reported as an important CBB bio-control agent, acting in conjunction with other species of ants to effectively suppress CBB at various life stages . Therefore, this technique could support Azteca ants as well as other ant species that play an important role in suppressing CBB populations. Our results support the general hypothesis that connectivity, one measure of habitat complexity, can sustain important ecological processes in natural and managed ecosystems. In aquatic systems, more complex habitats with macrophytes allow for greater food capture and maintain higher levels of diversity . In terrestrial systems, higher complexity can influence trophic dynamics . In coffee agroecosystems, ants are highly sensitive to habitat change and management intensification, generally expressed as the reduction of shade, elimination of epiphytes, and use of chemical inputs . Such intensification can have a negative effect not only on vegetation connectivity and ant foraging, but may also cascade to affect ecosystem services, such as biological control. Our study supports the idea that promoting complexity at a local scale, in this case providing structural resources for ants in agroecosystems, can significantly enhance connectivity within the arboreal strata, and potentially improve biological control of coffee pests. This idea has already been successfully implemented in other agricultural systems, placing “ant bridges” made of bamboo strips or strings connecting neighboring trees in , and could be incorporated as a management strategy in coffee systems. Future research should evaluate the practical feasibility of adding connections between vegetation strata to enhance bio-control. For example,studies in timber plantations have estimated that the presence of ants increases timber production by 40%, and that ants can be maintained at lower costs by providing intra-colony host tree connections using rope, poles or lianas . It is important that future studies in coffee also consider the costs of other CBB control methods, such as the application of the pesticide endosulfan, which can lead to the development of resistance, can negatively impact natural enemies, and can have harmful impacts on human health . Further investigation into promoting ant bio-control with artificial connections in coffee should: 1) assess economic tradeoffs, management applicability, and farmers’ perceptions of this method in large and small coffee plantations, 2) compare the cost between string placement and other management approaches , and 3) assess coffee yields on connected and not connected plants to provide management recommendations. More broadly, incorporating conservation bio-control strategies in combination with vegetation connectivity is consistent with criteria identified as key for the sustainability of biological control, such as increasing local habitat quality and enhancing species’ dispersal ability . Generally, the maintenance of shade trees and natural vegetation in agroforestry systems may increase vegetation complexity and natural connectivity between plants to promote ant foraging and subsequent biological pest control. The extent to which biotic and abiotic factors shape species interactions and their distributions has been a central focus for community ecologists . Included in the wide spectrum of factors shaping community dynamics are the diversity and availability of food resources and habitat complexity; all are important determinants of species diversity, distribution and interactions . Recent theoretical advances and empirical work in community ecology suggest that further research is needed to understand how resource availability and environmental factors shape species interactions. Specifically, one research gap is in our understanding of how variation in resources along habitat complexity gradients affects species interactions . However, empirically demonstrating how resources vary and how species respond is methodologically complicated, in the sense that the manipulation of multiple resources and consumers is not always straight forward, especially in complex systems. Consequently, studies have focused primarily on one single resource and– disproportionately– on competitive species interactions. Further, the environmental context in which resources matter is also not well understood and will gain more importance as natural and managed systems continue to experience habitat simplification .

Pleasant esters in red wines include ethyl acetate which has a OAV threshold of 12264 µg•L-1 and is described as fruity and balsamic , as well as isoamyl acetate, described as banana aroma with a OAV threshold of 30 µg•L-1 . In 2020, ethyl acetate was reduced in C0 and D5 wines, shading and reduced cluster temperatures preserved isoamyl acetate aromas in D1, D3 and D5 wines. When compared to wines from 2021, cooler vintage conditions did not result in ester compositional changes in exposed and shaded wines. Similarly, fatty acid esters were preserved in shaded wines, while 2020 C0 wines consistently had the lowest concentration of all measured fatty acid ethyl esters and various esters, all of which are associated with fruity and candy-like aromas. Concentrations of ethyl octanoate and ethyl decanoate remained beneath the reported perception threshold, thus observed shifts in composition with shading may be undetectable in Cabernet Sauvignon wines. However, ethyl hexanoate and ethyl isovalerate have remarkably low OAV thresholds of 5 µg•L-1 and 1 µg•L-1 , respectively . In the present study, all wines were above these thresholds, indicating that reductions in fruitiness may be perceived. This overall decrease in fruity aromas with cluster exposure and excess temperatures may negatively impact the marketability of Cabernet Sauvignon wines from hot viticulture regions with increasingly more frequent heat wave events associated with climate change.

Unpleasant and rancid aromas include isobutyric acid which imparts a cheese aroma and benzaldehyde which is associated with almond aroma in red wines . In this study, growing raspberries in container isobutyric acid concentrations were only affected in 2020, with D4 having the highest isobutyric acid concentration. The detection threshold for this aroma compound is 2300µg•L-1 . Concentrations detected in the experimental wines were substantially below this threshold, indicating that this slight increase in rancid aromas in D4 wines may not negatively impact overall wine perception. Given that D4 wines also exhibited enhanced fruitiness in with improved ester composition, the trade-off of slight increases in rancid aromas may be offset by the net benefit from increased fruity aromas in the wine aroma profile. While terpenes are often critical in white wines, these compounds when present in red wines have a large effect on wine aromas as their OAV thresholds are relatively low . The OAV threshold for a-terpinene, cis-rose-oxide and linalool are 250 µg•L-1 , 0.2 µg•L-1 and 25.2 µg•L-1 , respectively . The In 2020, α-terpinene, cis-rose-oxide and linalool were all reduced in C0 wines compared D4 and D5 wines, however concentrations of these compounds did not exceed the OAV threshold. These compounds produce odors such as peach, citrus, rose, and floral aromas in red wines . Previous work indicated an increase in terpenoids, particularly linalool in wines produced from fruit under black and red shade nets .

It was demonstrated that heat treatment will down regulate genes encoding key enzymes in terpenoid metabolism in Cabernet Sauvignon grapevines . Thus, increases in terpenoid content in shade film wines in 2020 may be due to reduced cluster temperature in a growing season with frequent heat wave events. In 2021, C0 wines exhibited the highest concentration α-terpinene, while cis-rose-oxide concentrations remained low in C0, and linalool was unaffected. In 2021, a cooler growing season with fewer days above 38°C may have resulted in less variation in terpenoid composition and net accumulation of terpenoids in exposed fruit . Ultimately, climatic shifts towards more frequent heat wave events will reduce floral and citrus aromas in wines produced from overexposed clusters. However, the year-to-year weather variation will enhance the unpredictability of the development of these compounds, leading to challenges for wine producers looking to produce a consistent product. As carotenoid breakdown products, C-13 norisoprenoids like β-damascenone often described by sweet and floral aromas and has an OAV threshold of 0.05 µg•L-1 . C-13 norisoprenoids have been shown to have a positive linear relationship with sunlight exposure to the grape cluster . Under extreme light intensity and temperature conditions, there are decreases in carotenoid concentration in the berry, thus reducing C-13 norisoprenoid precursors. In the present study, β- damascenone was highest in C0, D4 and D5 wines in 2020, while β-damascenone was highest in C0 and D1 wines in 2021, contrary to previous findings in hot viticultural areas. Lee et al. reported that grape clusters without leaf removal and inner canopy clusters contained more β- damascenone than south-facing clusters exposed to solar radiation by leaf removal. Likewise, black cloth and red shade net enhanced β-damascenone concentration compared to uncovered control .

Despite varied reports, Lee et al. also demonstrated a linear positive relationship between norisoprenoids in the grape berry and concentrations in wine, with the berry concentration was always greater than that of the resultant wines. It may be possible that carotenoid degradation due to excessive temperatures in C0 treatments was negligible or less than the biosynthesis of C-13 norisoprenoids, resulting in similar concentrations as D4 and D5 shade film treatments. Therefore, the results of this study demonstrated that partial solar radiation exclusion with reductions in UVA, UVB and NIR radiation does not hinder norisoprenoid content in wines. Additionally, the concentrations of β- damascenone across all treatments in both years exceeded the odor active threshold for this compound, indicating that significant differences in β-damascenone concentrations between C0 and treatments may be perceivable in resultant wines.Although grapevine is one of the most resilient crops globally, the changing climate is one of the many challenges to its cultivation and ultimately wine production. According to the Intergovernmental Panel on Climate Change , global air temperatures are likely to increase 1.5-4.5o C between 2030 and 2052 . Historical records of growing degree day accumulation demonstrate that air temperatures in the world’s most notable grape growing regions have increased steadily within the last 70 years . Such increases in GDD accumulation disrupt the natural coupling and balance of primary and secondary metabolites during ripening, corresponding with a plateau in wine quality ratings . Subsequently, excessive air temperatures reduce pleasant and desirable wine aroma compounds, but also contribute to reductions in wine quality . Moreover, predicted changes in precipitation patterns and increased drought frequency threaten grapevine water status conducive to market-desired fruit yield and composition and long-term vineyard sustainability. With increasingly frequent and prolonged drought periods and less predictable rainfall, vineyards will need to adapt to changes in water availability. The evaluation and performance of drought tolerant root stock and scion cultivars has also been investigated ; however, their adoption may be limited due to historical and cultural connections to popular grape cultivars. Additionally, the effect of root stock-scion interactions on water use efficiency is poorly understood and only recently became an area of research . At the field scale, large plastic pots for plants wider row spacing to decrease vine density can decrease overall vineyard evapotranspiration by reducing the competition for water between vertically shoot-positioned vines . Pieri et al. examined the water balance of two planting densities in five viticultural regions of France and concluded that lower planting densities utilizing constrained canopies can maintain vine water status within moderate stress levels in forecasted climate change conditions. However, decreased plant density may result in lower yields at the field-scale, requiring an economic cost/benefit analysis to determine its viability as a water conserving solution. Reducing applied water through sustained and regulated deficit irrigation strategies is already commonly used to improve flavonoid composition in the grape skins . Furthermore, varied amounts of applied water have been trialed in a hot growing region such as California and demonstrated that irrigating at 50% of potential grapevine evapotranspiration was sufficient to mitigate water shortages when dormant season precipitation was limited . More deliberate interventions to adapt to heat and water shortages in the vineyard include the use of shade cloths and films to reduce canopy temperatures and vine evapotranspiration . Again however, the implementation of these shade structures is under question as they present barriers to vineyard mechanization and may be a costly and unfeasible long-term solution.

Adapting trellis systems may be another method for mitigation of climate change impacts in production vineyards. Choosing an appropriate trellis system is an important pre-planting decision during vineyard establishment. An appropriate trellis system optimizes the vine’s capacity to intercept solar radiation and produce a canopy microclimate that results in optimal berry ripening without excessive direct solar radiation overexposure to the fruit zone. A traditional and commonly used trellis system in various grapevine production areas worldwide is the vertically shoot positioned trellis. While VSP trellises were traditionally thought to improve berry ripening, the VSP trellis system maximizes light penetration and canopy porosity, producing a canopy microclimate which increases cluster vulnerability to overexposure in hot viticulture regions . More recent work has investigated the resiliency of red wine grapes in hot viticulture regions when training systems are varied from the traditional VSP trellis. In a study conducted in Napa Valley CA, USA, trellis systems with free and sprawling canopies such as a single-high wire and high-quadrilateral systems increased yield and produced berries with improved flavonoid profiles that are attributed to reduction in chemical degradation compared to traditional VSP trellis systems regardless of applied water amounts . Additionally, the interactive effect of varied applied water amounts and trellis systems has been minimally investigated. Williams and Heymann applied various fractions of estimated potential grapevine evapotranspiration to VSP and Scott-Henry trellis systems to elucidate the effect of applied water on vine productivity and fruit composition in Livermore, CA. In their study, irrigation amounts had a larger effect on vine productivity and berry quality than trellis systems due to VSP and Scott-Henry trellis systems having similar levels of overexposure to the fruit zone . Free and sprawling trellis systems which can shade the fruit zone and protect it from overexposure conditions can provide a long-term feasible heat avoidance strategy for hot viticulture regions due to promoting larger canopies. While there are demonstrated improvements to grape chemical composition with the adoption of these sprawling trellis systems, it is understood that trellis systems promoting larger leaf area indices will have a higher water demands . In regions where irrigation is required to supplement seasonal precipitation, maintaining these larger canopies may prove difficult, especially with increasingly stringent environmental regulations such as the Sustainable Groundwater Management Act in the state of California. SGMA limits groundwater extraction for agricultural irrigation . Compliance with SGMA will result in allotted water use restrictions in California’s Napa Valley, limiting growers to 120 mm of applied water each season. Therefore, there is uncertainty surrounding how growers will respond to such water restrictions in tandem with adapting to increasing temperatures in a climate change scenario. Ultimately, there is a lack of information on the water footprint of resilient trellis systems hindering their adoption in microirrigated wine grape production vineyard. Our previous works conducted with VSP trellis indicated that irrigating at different percent of potential grapevine ET affected grapevine physiology leading to different carbon allocation, water footprint, and water use efficiency in hot climates . Likewise, our previous work provided evidence that trellis systems other than VSPs provide better adaptation of wine grapes to climate change by ameliorating physiological performance and berry chemistry due their canopy architecture . Therefore, the objective of this experiment was to determine the water use efficiency of grapevine with trellis and pruning types that are commonly used in production regions characterized by hot climates. We hypothesized that the new trellises that were indicated to be more resilient to climate change would have different water needs than traditional VSP. Therefore, we applied fractions of the potential grapevine evapotranspiration estimated for VSP to different trellis systems to compare their water productivity relative to VSP under a climate change scenario.Climate change in hot viticultural regions brings two prominent challenges: higher air temperatures with unpredictable heat wave events and increased drought frequency . At the experimental site in Oakville, CA, the 2021 growing season was cooler than both the preceding and following growing seasons. Hot air temperatures may create untoward growing conditions that affect whole grapevine physiology . In hot viticultural regions like California, prolonged drought conditions are becoming more common and increasingly severe. In the current study, extreme and exceptional drought conditions were recorded across the three experimental years. Total precipitation at the experimental site during the water year within the last decade was previously reported as approximately 768mm . The total precipitation recorded during the 2019-2020 and 2020-2021 growing seasons was 234.2mm and 278.3mm, respectively and was substantially less than the long-term average.

This aspect could have an important influence in the eligibility characteristics such as polyphenol composition. In fact, the content of polyphenols in grapes is clearly affected by four agro-ecological factors: the cultivar, the year of production , the site of production , and the degree of maturation. Moreover, the methods of vinification and applied technological procedures can significantly modify both the concentration and composition of polyphenols and, therefore, also the color intensity and hue of red wines. The increasing of yield and vine vigor can also affect the color, polyphenol composition, and sensory attributes of Cabernet Sauvignon wines. In conclusion, Sangiovese wines from Italy and California showed several significant chemical differences in term of eligible and identity profiles , such as polyphenols composition and volatiles that not completely affected the intrinsic sensory quality. Concerning the perceived quality, despite the Tuscan experts perceived differences between the Californian and Italian wines, they considered them similar when they evaluated their typicality. Finally, the results from this study confirm that perceived quality in terms of typicality of Sangiovese was still related not only to fruity and floral flavors but also to lightness and freshness, plastic pots for planting despite the intrinsic quality aspect of the “structure” of the wine and to what is considered a “good” color.

Moreover, the findings confirm that Sangiovese shows a flexibility in terms of chemical and sensory modification, according to the production area and that it can be considered typical even if it comes from an area far away from the traditional ones. Agriculture is a key human activity in terms of food production, economic importance and impact on the global carbon cycle. As the human population heads toward 9 billion or beyond by 2050, there is an acute need to balance agricultural output with its impact on the environment, especially in terms of greenhouse gas production. An evolving set of tools, approaches and metrics are being employed under the term “climate smart agriculture” to help—from small and industrial scale growers to local and national policy setters—develop techniques at all levels and find solutions that strike that production-environment balance and promote various ecosystem services. California epitomizes the agriculture-climate challenge, as well as its opportunities. As the United States’ largest agricultural producing state agriculture also accounted for approximately 8% of California’s greenhouse gas emissions statewide for the period 2000–2013. At the same time, California is at the forefront of innovative approaches to CSA. Given the state’s Mediterranean climate, part of an integrated CSA strategy will likely include perennial crops, such as winegrapes, that have a high market value and store C long term in woody biomass. Economically, wine production and retail represents an important contribution to California’s economy, generating $61.5 billion in annual economic impact.

In terms of land use, 230,000 ha in California are managed for wine production, with 4.2 million tons of winegrapes harvested annually with an approximate $3.2 billion farm gate value. This high level of production has come with some environmental costs, however, with degradation of native habitats, impacts to wildlife, and over abstraction of water resources . Although many economic and environmental impacts of wine production systems are actively being quantified, and while there is increasing scientific interest in the carbon footprint of vineyard management activities, efforts to quantify C capture and storage in annual and perennial biomass remain less well-examined. Studies from Mediterranean climates have focused mostly on C cycle processes in annual agroecosystems or natural systems. Related studies have investigated sources of GHGs, on-site energy balance, water use and potential impacts of climate change on productivity and the distribution of grape production. The perennial nature and extent of vineyard agroecosystems have brought increasing interest from growers and the public sector to reduce the GHG footprint associated with wine production. The ongoing development of carbon accounting protocols within the international wine industry reflects the increased attention that industry and consumers are putting on GHG emissions and offsets. In principle, an easy-to-use, wine industry specific, GHG protocol would measure the carbon footprints of winery and vineyard operations of all sizes. However, such footprint assessment protocols remain poorly parameterized, especially those requiring time-consuming empirical methods.

Data collected from the field, such as vine biomass, cover crop biomass, and soil carbon storage capacity are difficult to obtain and remain sparse, and thus limit the further development of carbon accounting in the wine sector. Simple yet accurate methods are needed to allow vineyard managers to measure C stocks in situ and thereby better parameterize carbon accounting protocols. Not only would removing this data bottleneck encourage broader participation in such activities, it would also provide a reliable means to reward climate smart agriculture.Building on research that has used empirical data to compare soil and above ground C stocks in vineyards and adjacent oak woodlands in California, this study sought to estimate the C composition of a vine, including the relative contributions of its component parts . By identifying the allometric relationships among trunk diameter, plant height, and other vine dimensions, growers could utilize a reliable mechanism for translating vine architecture and biomass into C estimates . In both natural and agricultural ecosystems, several studies have been performed using allometric equations in order to estimate above ground biomass to assess potential for C sequestration. For example, functional relationships between the ground-measured Lorey’s height and above ground biomass were derived from allometric equations in forests throughout the tropic. Similarly, functional relationships have been found in tropical agriculture for above ground, below ground, and field margin biomass and C. In the vineyard setting, however, horticultural intervention and annual pruning constrain the size and shape of vines making existing allometric relationships less meaningful, though it is likely that simple physical measurements could readily estimate above ground biomass. To date, most studies on C sequestration in vineyards have been focused on soil C as sinks and some attempts to quantify biomass C stocks have been carried out in both agricultural and natural systems. In vineyards, studies in California in the late 1990s have reported net primary productivity or total biomass values between 550 g C m−2 and 1100 g C m−2. In terms of spatial distribution, some data of standing biomass collected by Kroodsma et al. from companies that remove trees and vines in California yielded values of 1.0–1.3 Mg C ha−1  year−1 woody C for nuts and stone fruit species, and 0.2–0.4 Mg C ha−1  year−1 for vineyards. It has been reported that mature California orchard crops allocate, on average, one third of their NPP to the harvested portion and mature vines 35–50% of the current year’s production to grape clusters. Pruning weight has also been quantified by two direct measurements which estimated 2.5 Mg of pruned biomass per ha for both almonds and vineyards. The incorporation of trees or shrubs in agroforestry systems can increase the amount of carbon sequestered compared to a monoculture field of crop plants or pasture. Additional forest planting would be needed to offset current net annual loss of above ground C, representing an opportunity for viticulture to incorporate the surrounding woodlands into the system. A study assessing C storage in California vineyards found that on average, drainage for plants in pots surrounding forested wildlands had 12 times more above ground woody C than vineyards and even the largest vines had only about one-fourth of the woody biomass per ha of the adjacent wooded wildlands.The objectives of this study were to: measure standing vine biomass and calculate C stocks in Cabernet Sauvignon vines by field sampling the major biomass fractions ; calculate C fractions in berry clusters to assess C mass that could be returned to the vineyard from the winery in the form of rachis and pomace; determine proportion of perennially sequestered and annually produced C stocks using easy to measure physical vine properties ; and develop allometric relationships to provide growers and land managers with a method to rapidly assess vineyard C stocks.

Lastly, we validate block level estimates of C with volumetric measurements of vine biomass generated during vineyard removal.The study site is located in southern Sacramento County, California, USA , and the vineyard is part of a property annexed into a seasonal floodplain restoration program, which has since removed the levee preventing seasonal flooding. The ensuing vineyard removal allowed destructive sampling for biomass measurements and subsequent C quantification. The vineyard is considered part of the Cosumnes River appellation within the Lodi American Viticultural Area, a region characterized by its Mediterranean climate— cool wet winters and warm dry summers—and by nearby Sacramento-San Joaquin Delta breezes that moderate peak summer temperatures compared to areas north and south of this location. The study site is characterized by a mean summer maximum air temperature of 32 °C, has an annual average precipitation of 90 mm, typically all received as rain from November to April. During summer time, the daily high air temperatures average 24 °C, and daily lows average 10 °C. Winter temperatures range from an average low 5 °C to average high 15 °C. Total heating degree days for the site are approximately 3420 and the frost-free season is approximately 360 days annually. Similar to other vineyards in the Lodi region, the site is situated on an extensive alluvial terrace landform formed by Sierra Nevada out wash with a San Joaquin Series soil . This soil-landform relationship is extensive, covering approximately 160,000 ha across the eastern Central Valley and it is used extensively for winegrape production. The dominant soil texture is clay loam with some sandy clay loam sectors; mean soil C content, based on three characteristic grab samples processed by the UC Davis Analytical Lab, in the upper 8 cm was 1.35% and in the lower 8–15 cm was 1.1% . The vineyard plot consisted of 7.5 ha of Cabernet Sauvignon vines, planted in 1996 at a density of 1631 plants ha−1 with flood irrigation during spring and summer seasons. The vines were trained using a quadrilateral trellis system with two parallel cordons and a modified Double Geneva Curtain structure attached to T-posts . Atypically, these vines were not grafted to rootstock, which is used often in the region to modify vigor or limit disease .In Sept.–Oct. of 2011, above ground biomass was measured from 72 vines. The vineyard was divided equally in twelve randomly assigned blocks, and six individual vines from each block were processed into major biomass categories of leaf, fruit, cane and trunk plus cordon . Grape berry clusters were collected in buckets, with fruit separated and weighed fresh in the field. Leaves and canes were collected separately in burlap sacks, and the trunks and cordons were tagged. Biomass was transported off site to partially air dry on wire racks and then fully dried in large ventilated ovens. Plant tissues were dried at 60 °C for 48 h and then ground to pass through a 250 μm mesh sieve using a Thomas Wiley® Mini-Mill . Total C in plant tissues was analyzed using a PDZ Europa ANCA-GSL elemental analyzer at the UC Davis Stable Isotope Facility. For cluster and berry C estimations, grape clusters were randomly selected from all repetitions. Berries were removed from cluster rachis. While the berries were frozen, the seeds and skins were separated from the fruit flesh or “pulp”, and combined with the juice . The rachis, skins and seeds were dried in oven and weighed. The pulp was separated from the juice + pulp with vacuum filtration using a pre-weighed Q2 filter paper . The largest portion of grape juice soluble solids are sugars. Sugars were measured at 25% using a Refractometer PAL-1 . The C content of sugar was calculated at 42% using the formula of sucrose. Below ground biomass was measured by pneumatically excavating the root system with compressed air applied at 0.7 Mpa for three of the 12 sampling blocks, exposing two vines each in 8 m3 pits. The soil was prewetted prior to excavation to facilitate removal and minimize root damage. A root restricting duripan, common in this soil, provided an effective rooting depth of about 40 cm at this site with only 5–10 fine and small roots able to penetrate below this depth in each plot. Roots were washed, cut into smaller segments and separated into four size classes , oven-dried at 60 °C for 48 h and weighed. Larger roots were left in the oven for 4 days.

Meta-analysis of cohort studies has identified increased risk in obese individuals =1.89, 95% confidence interval : 1.51–2.36 for developing liver cancer compared to normal weight individual while meta-analysis of prospective studies correlated a larger waist circumference and waist–hip ratio with increased risk for colon cancer. Similarly, diabetics are more than twice as likely to be diagnosed with cancer of the liver, pancreas, endometrium, and to a lesser extent, colon, breast, and bladder.5 Many possible underlying mechanisms including hyperinsulinemia, hyperglycemia, and inflammation have been proposed to explain the increased cancer incidence. Insulin, produced by b-cells in the pancreas, is released to promote cellular absorption of blood glucose and many factors including excess weight and increased plasma triglyceride levels can raise circulating levels of insulin. Chronic insulin elevation results in resistance, which then increases the biological activity of insulin-like growth factor , an endocrine and paracrine hormone regulating tissue growth and metabolism. Epidemiological studies have linked IGF-1 to several cancer types, including hepatocellular carcinoma and colorectal cancer . Moreover, excess adiposity leads to the derangement of other peptide hormones such as resistin, leptin, adiponectin, pot with drainge holes and tumor necrosis factor α , contributing to the metabolic abnormalities commonly observed in obese and diabetic individuals.

Indeed, increased leptin and reduced adiponectin have been identified as risk factors for the progression of liver steatosis, fibrosis, and tumorigenesis as well as CRC formation. Both bile acids and the intestinal microbiota have been extensively studied in the context of various health conditions, particularly obesity and type II diabetes-associated HCC and CRC. Although the exact mechanism of how gut microbes and BAs affect one another remains unclear, it is evident that the introduction of intestinal microbes increased liver cholesterol and altered BA profiles in germ-free mice. Conversely, dietary BA supplementation can modulate gut microbial profile in animal models.This review will focus on the current understanding of the complex interplay between BAhomeostasis and gut microbial profiles in regards to obesity and diabetes-associated liver and colon carcinogenesis. They are synthesized by cholesterol catabolism in the liver through both the classical and the acidic pathway , which differ in the modification order of the sterol ring and side chain oxidation. BAs synthesized in the liver are subsequently conjugated for storage in the gallbladder.

Upon ingestion of fat and protein, cholecystokinin, a peptide hormone in the small intestine will stimulate the release of bile containing digestive enzymes and primary BAs, cholic acid and chenodeoxycholic acid , from the gall bladder. These BAs will then activate farnesoid x receptor in the liver which induces the expression of small heterodimer partner to inhibit the activity of liver receptor homolog-1 responsible for upregulating the rate-limiting BA synthesis enzyme CYP7A. Intestinal FXR activity on the other hand, induces the expression of fibroblast growth factor 19 which binds hepatic fibroblast growth factor receptor 4 and activates c-Jun N-terminal kinase 1/2 and extracellular signal-regulated kinase 1/2 to inhibit BA synthesis. BAs are actively reabsorbed from the ileum by the ileal BA transporters and circulated back to the liver through the hepatic portal vein. This highly efficient process ensures that a majority of synthesized BAs are recycled with only 1-2% being converted into secondary BAs, deoxycholic acid , and lithocholic acid , by bacterial 7α-dehydroxylation in the terminal ileum and colon and excreted in feces. The regulation of BA circulation between the liver and intestines is summarized in Figure 1. In addition to modulating BA synthesis, FXR also regulates the expression of several transporters including apical sodium dependent BA transporter , fatty acidbinding protein subclass 6, and organic solute transporter α and β to control the absorption of not only BAs but also lipids, vitamins, and xenobiotics. Interestingly, recent studies have implicated FXR in the interplay between obesity-associated BA dysregulation and gut dysbiosis to potentially promote carcinogenesis in the liver and colon. Within the context of obesity and diabetes, FXR can regulate glucose and lipid homeostasis through actions at various sites along the gut-liver axis.

Agonist activation or hepatic overexpression of FXR significantly lowered blood glucose levels in both diabetic and wild type mice. FXR stimulation also decreased blood low density lipoprotein levels and inhibited fatty acid and TG synthesis in mice fed a high sugar and fat diet. These combined effects of FXR protected mice against body weight gain, liver and muscle fat deposition, and reversed insulin resistance. At the metabolic level, FXR functions to repress hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis genes. Consequently, FXR stimulation promotes glycogen synthesis and enhances insulin sensitivity in obese mice. FXR activity in pancreatic β-cell lines and human islets can regulate transcription factor Kruppellike factor 11 to increase insulin gene expression and protein kinase B-dependent phosphorylation and translocation of glucose transporter 2 at the plasma membrane of hepatocytes. By stimulating pancreatic insulin secretion and hepatic glucose uptake, FXR can effectively delay the pathological progression of insulin resistance, hyperglycemia, and glucosuria in diabetic mice. Consistently, FXR knockout mice exhibited glucose intolerance, insulin insensitivity, elevated serum TG, cholesterol, and BA levels resulting in greater hepatic fat accumulation compared to wild type mice. Hepatic FXR expression is conversely regulated by glucose levels in streptozotocininduced diabetic rats. Chromatin immunoprecipitation in mice also revealed that long-term high-glucose exposure increased histone acetylation and demethylation on the FXR-target Cyp7a1 gene promoter region leading to elevated basal expression and consequently, a larger BA pool with altered composition. These observations strongly support the existence of crosstalk between the cellular mechanisms regulating glucose, lipid, and BA homeostasis in the liver and intestines with FXR serving mediator. A link between FXR and enterohepatic cancer was firmly established when FXR KO mice were found to have markedly elevated hepatic inflammatory and oxidative stress markers compared to WT mice and a striking 100% incidence rate of spontaneous liver tumors between 13 and 15 months of age. BA-containing diet further exacerbated inflammation and oxidative stress in FXR KO mouse liver supporting that BA dysregulation subjects hepatocytes to higher oxidative stress. Hepatocyte-specific over expression of SHP failed to alter liver tumor incidence or size in FXR KO mice, large pot with drainage but did result in lower neoplasia grade, decreased cell proliferation, and increased apoptosis. Moreover, FXR stimulation can down-regulate lipopolysaccharide -induced, nuclear factor kappa-light-chainenhancer of activated B cells -mediated hepatic inflammation by suppressing the expression of proinflammatory mediators in human HCC cells and mouse primary hepatocytes. FXR KO mice displayed higher hepatic mRNA levels of inducible nitric oxide synthase, prostaglandin-endoperoxide synthase 2 , chemokine ligand 10, and interferon type II which resulted in exaggerated inflammation and necrosis after LPS exposure at a dose that failed to elicit measurable liver injury or inflammation in WT mice. The HCC in FXR KO mice was associated with sustained oncogenic Wnt/B-catenin signaling through Wnt4 and disheveled induction, E-cadherin repression, and glycogen synthase kinase-3B inactivation as the mice aged. Furthermore, microarray analysis of FXR KO mouse liver revealed altered gene expression profiles related to metabolism, inflammation, and fibrosis compared to WT liver recapitulating human HCC progression. Liver tumor bearing FXR KO mice showed elevated levels of interleukin 6 and signal transducer and activator of transcription 3 due to diminished expression of suppressor of cytokine signaling 3, a direct FXR target gene. STAT3 activation in conjunction with elevated TNFα and IL-6 levels has been shown to potentiate HCC formation.

Additionally, FXR can epigenetically silence the promoter of gankyrin, a proteasome subunit responsible for the degradation of retinoblastoma, p53, hepatic nuclear factor 4 alpha, and CCAAT /enhancer-binding tumor suppressor proteins. The loss of FXR in mice increased gankyrin expression to promote tumorigenesis. Interestingly, long-lived little mice with high basal FXR expression do not develop liver cancer with age or carcinogen administration due to insufficient gankyrin induction. FXR activation in human hepatocytes and hepatoma cells protected against cytotoxicity induced by cisplatin and other DNA-damaging agents. These findings support that in addition to its metabolic regulation, FXR also functions to modulate oxidative stress, inflammation, and cell proliferation to inhibit cancer development. Evidence also exists to suggest that FXR may act as a modulator of intestinal inflammation and a link between BA homeostasis and the intestinal microbiome. In the small intestine, FXR negatively regulates the expression of transporters involved in BA reabsorption while inducing the production and secretion of FGF19/Fgf15 to inhibit hepatic BA synthesis. Colon inflammation in Crohn’s disease patients and rodent colitis models is correlated with reduced FXR mRNA levels. The progression of colon inflammation is exacerbated in FXR KO mice while treatment with FXR agonist attenuated colonic tissue damage and immune cell activation. Conversely, FXR stimulation protected WT mice from chemical-induced colitis by reducing epithelial permeability, ulceration, and inflammatory cell infiltration. Moreover, FXR agonist-treated WT mice and differentiated enterocyte-like cells displayed lower pro-inflammatory cytokines and better preserved epithelial barrier function. In addition to its beneficial effects on intestinal function and inflammation, a connection between FXR and intestinal microbes was observed when ampicillin-treated mice had inhibited ileal expression of FXR, SHP, and FGF19/Fgf15. Expression of FXR and its target genes levels were rescued by combination treatment with CA, but not taurocholic acid, in ampicillin-treated mice suggesting that enterobacteria can enhance BA-mediated FXR activity via taurocholic acid deconjugation. Furthermore, intestinal inflammation in mice down-regulated FXR expression in a toll-like receptor 9 -dependent manner since the FXR promoter contains a response element to interferon regulatory factor 7, a TLR9- regulated factor. These preliminary findings suggest a possible role of intestinal FXR as a mediator between BA homeostasis, the gut microbiome, and host immunity to prevent excessive inflammation and maintain GI health. Examination of FXR in human HCC samples and cell lines has yielded further evidence to support its protective role against cancer formation. Marked reduction in FXR levels and activity were observed in human HCC samples compared to normal liver tissue. This reduction resulted from inhibition of hepatic nuclear factor 1 alpha activity on the FXR genepromoter by elevated pro-inflammatory mediators. The 3′ untranslated region of FXR mRNA was found to be a target of miR-421 and FXR downregulation by miR-421 promoted proliferation, migration, and invasion in human HCC cells. Decreased FXR levels in HCC cells also correlated with overexpression of active Ras resulting in strong activation of ERK1/2, a common characteristic of malignant cells. However, additional studies are required to determine whether dysregulated FXR activity increases the risk of HCC and CRC. One possible underlying cause of FXR insufficiency in humans is genetic variation in the gene itself resulting in diminished expression or function. Indeed, sequencing analysis of FXR in intrahepatic cholestasis of pregnancy patients revealed four functional heterozygous variants, three of which demonstrated functional defects in either translation efficiency or signaling activity. Additionally, FXR polymorphism identification analysis of European-, African-, Chinese-, and Hispanic-Americans identified a common, hypomorphic single nucleotide polymorphism with population allelic frequencies ranging from 2.5% to 12.1% . The in vitro transactivation activity of this hypomorphic SNP was lower relative to that of WT allele and human carriers of this allele showed significantly reduced hepatic SHP levels. Furthermore, the global FXR haplotype distribution between inflammatory bowel disease and healthy individuals was significantly different which emphasizes the link between FXR-mediated BA signaling and intestinal inflammation.48 Since chronic inflammation is widely considered a predisposition to cancer development, enhancement of FXR signaling appears to be a promising clinical target to not only normalize the BA dysregulation seen in obese and diabetic individuals but also combat chronic hepatic and intestinal inflammation. The appropriate circulation of BAs between the liver and small intestine is crucial to the maintenance of BA homeostasis and consequently, normal GI physiology. The ileum is where approximately 90% of secreted BAs are actively reabsorbed into the bloodstream by ASBT for transport back to the liver through the hepatic portal vein. Because of its predominantly ileal expression and central role in enterohepatic cycling of BAs, ASBT is another potential participant in the interplay between BA dysregulation and gut dysbiosis. In Caco-2 cells, 25-hydroxycholesterol and CDCA treatments greatly reduced ASBT promoter activity and mRNA levels through the actions of FXR, SHP, retinoic acid receptor, and retinoid x receptor . Mice fed a cholesterol-enriched diet exhibited downregulation of ASBT at both the mRNA and protein levels, decreased ileal BA uptake, and elevated fecal BA excretion. Interestingly, exposure of Caco-2 cells to pro-inflammatory factor IL-1B also caused a 65% reduction in ASBT mRNA level. Elevated levels of cholesterol in the intestinal lumen and pro-inflammatory mediators in the intestinal epithelium appear to down-regulate ASBT activity, thereby disrupting enterohepatic BA circulation.

Clusters were chosen from the most basal position on the shoot arising from the most basal bud. Flowering was estimated by percent of the cluster appearing to have caps fallen and flowers showing. At the point where 50% of the caps on flowers have fallen away, called anthesis, the cluster is considered at full bloom . Flowering is recorded as a percent estimate of the cluster in bloom. These clusters were then tagged with fluorescent tape loosely and followed subsequently for veraison and maturity. Records were taken every two to three days during each of the phenological stages. At each stage of monitoring, researchers calibrate observations with each other and with photographs from previous years. The Wang and Engel model uses a maximum temperature of 40°C, which may be the biological threshold for grapevine growth . The biological optimal temperature for grapevines is likely around 25°C , and when temperatures exceed 30°C, there are impacts on anthocyanins , and temperatures exceeding 37°C decreased coloration in grape berry skin and degradation of aromatic compounds .

Therefore, we include the variable of cumulative number of days when temperature reached a maximum at or over 40°C in our models of veraison, the only phenological stage that encounters these days, round pot with variety as a random effect . This study spanned four years and is ongoing to evaluate the sensitivity of different varieties to climate change. It is important to note that timing and duration of the winter pruning was variable, which could have introduced error because the timing of pruning can impact budburst . Some varieties were discontinued from the study because of death, disease, or pest damage. Some varieties were only included in later years of the study once they reached maturation. Measurements of percent budburst, flowering, and veraison were converted to GDD using R . For each stage, a linear model was fit with phenological development as a percent as the response variable and time as the independent variable. The fitted model was used to estimate the day a cluster reached 50% budburst, flowering, or veraison. Data were cleaned by removing individuals with illogical estimates for timing. It was determined for these removed points that too few measurements were made for those vines, and the individuals removed were from the year 2015. The limits for estimates were based on observational data. If budburst was predicted earlier than day 60 of the year, this individual was removed, because this was earlier than measurements were recorded.

Flowering was limited to day 111 of the year, and veraison was limited to begin at day 175 of the year. We obtained for each of the four years the GDD’s required to reach the three phenophases for an individual plant. These GDD’s were the response variable of the hierarchical models described in the next section. The days over 40°C from January until September were also quantified for each year. To quantify the variation in growing degree days across and within grapevine varieties, we used Bayesian linear mixed effect models as implemented in the package RStanArm . The default set of priors was used for the RStanArm package. A model using GDD’s with a base temperature of 10°C were compared against a model with base 0°C temperature. Models were fit using GDD with a base temperature of 0°C on the day of a phenological event as the response variable. Separate models were fit for the three phenological stages: budburst, flowering, and veraison. For the budburst and flowering stages, GDD was the response variable with “utility” as a fixed effect and “geography/variety” as random effects. For veraison, we included “utility” as a fixed effect, “geography/variety” as random, as well as “days above 40°C” as a random effect tied to variety. The climate data were summarized in R from the raw CIMIS data, and the cumulative measurements for each stage included the weeks prior to each stage. The explanatory models of maximum daily temperature, and cumulative number of days with temperatures reaching over 40°C, and year as fixed effects were incorporated into models for each stage. Overall, we added grape utility, in terms of wine or table grape or both, geographic groups, and country of origin as nested random variables .

The advantage to using a fixed effect model to predict GDD by variety is that we fill gaps by including data from other varieties’ responses to predict individual variety response. We quantify variety-level GDD by leveraging information from all varieties together. Rather than using an average for each variety by year, we utilize the temporal redundancy to estimate consistency across years, and we can see from the average of all varieties together which ones fluctuate the most during years with more change in climate. We used the posterior predictive check of a PSIS diagnostic plot to ensure khats were all less than 0.7. The preference for rstanarm to evaluate these mixed effects models is based on the Bayesian approach using MCMC, rather than restricted maximum likelihood estimation, which tends to underestimate uncertainties . This Bayesian approach estimates uncertainty for all the model levels, including our random effect of variety which contains 137 or less estimated parameters. When ELPD-difference was compared, the differences in log probability for the five models were almost completely within their individual standard errors. For this reason, we can consider all five models substantially predictive, but we chose the top model based on lowest ELPD-difference and biological relevance of the variables in the model. For veraison, the differences in log probability , were not within their individual standard errors . The models of veraison were improved by the addition of the variable, “days above 40,” referring to the cumulative number of days with daily maximum temperatures at or above 40°C. The genetically identified geographic origins provided by Bacilieri et al. Supplemental Information added predictive information to our final models for budburst, flowering, and veraison. We see differences in sensitivity to climate across stages for each of the geographic groups, visualized by the coefficient of variation over the four years analyzed . The intercepts reported in Supplementary Table 2, in terms of growing degree days, provide a predictive range to expect phenological variability from these groups. The range of phenological timing for specific cultivars can help match varieties with ideal climates and regions. There are varieties from each of these regions with the potential to be late ripening. From the Italian Peninsula, there is Dolcetto with a relatively early veraison and Aglicanico with a relatively late and variable veraison . The timing of stages can be extremely consistent, such as with Gamay Noir from Western Central Europe, but there can also be a wider range of timing like that of Mourvedre, from the same region. Therefore, while region is predictive, analyzing the timing for specific varieties is also useful when selecting alternative varieties for planting. years. Budburst had the highest coefficient of variation, likely due to the impact of conditions during dormancy . There may also be an accumulation of climatic impacts over the season resulting in the highest variability in timing at veraison. In a previous common garden experiment, round plastic planter the timing of maturity also had the largest standard error with more predictable timing for budburst and flowering . Sensitivity across stages does not have a strong correlation, but Budburst and Flowering seem to have the strongest relationship, with the highest R2 for the Balkans geographic region at 96% . The parameter estimates of the three models reported the highest sigma for variety for all three models. From an ecological perspective, a vineyard is a system that responds to its environment. This system includes the soil, international varieties, and the climate. We modelled the response of varieties’ phenological timing to climate, and the results present unique sensitivities to climate over 4 years.

Geographic origin and cultivated utility of grapes explain some of the variation seen in phenological timing, which we expect is driven by physiological differences. Accumulation of daily temperature in our model is strongly correlated with phenological stage occurrence, which agrees with past modelling of growing degree days and phenology . Previous models have used individual parameters for growing degree days and base temperature based on the cultivar . Duchêne et al. used daily maximum temperature rather than GDD in their models to predict phenology, and their models included a stage specific base temperature. Our model is unique by including variety specific response to cumulative days above 40°C. We expected this to impact the timing of veraison for some varieties with higher sensitivity to heat stress. The chosen model for veraison included the variable of “days above 40°C,” which is in part due to the timing of high heat days, typically occurring later in the season, during this stage. This model outranked models for veraison that nested geographic origin, indicating that the effect of high temperature is not variety specific. The general intercepts of the models for each stage predict the mean GDD required to reach each phenological stage, and the intercepts for each variety indicate the specific GDD requirement for each variety . The general intercept for the three stages was 199 GDD for budburst, 836 GDD for flowering, and 1,699 GDD for veraison . We may expect for other regions and in California’s future that heat stress may impact flowering as we see an increase in high heat events earlier in the growing season . The dominant hypotheses indicate that budburst may be less correlated with growing season temperature changes because it is more impacted by viticultural techniques and therefore sensitive to chilling time over dormancy . In the UC Davis ampelography vineyard, all vines are experiencing the same dormancy conditions, so the difference within years in timing of budbreak is explained by the varietal differences . However, across years, the lower sensitivity of budburst timing compared to flowering and veraison may be also be explained in part by the discrepancy in the dominance of climatic versus genetic controls for vegetative versus reproductive growth, respectively . Varieties may not be sensitive to temperature in the same way across stages, as the vine switches from vegetative growth to reproductive growth with the onset of flowering . The weaker relationship between budburst and cumulative temperature than the subsequent stages may be because flowering time and maturation are more strongly controlled by genetics . Furthermore, the dissociation between vegetative and reproductive growth makes it unclear how plants will adapt to climate change . While research shows viticulture is expanding to new territories all over the world , a crucial aspect to the success of the expanding viticulture into novel territories is matching the phenology to the local climate; agriculture will fail when introduced crops cannot adjust to new seasons . Climate change will not only change the varieties suitable for a region, but also the regions suitable for planting grapes . Failure to choose appropriate varieties for novel territories can impact natural ecosystems, an unintended adverse effect of expanding viticulture . A recent study modeling changes in viticulture territories under climate change scenarios predicted that 51 % of climatically suitable for growing winegrapes would become unsuitable . The intraspecific variation in heat thresholds for grapevines impacts the adaptation capacity of each cultivar . Previous authors suggest allowing cultivar turnover to prevent these major losses, which will depend heavily on what governments allow in Europe , while we are free to plant many different varieties in California. Among many strategies of adaptation to climate change, shifting to climatically more appropriate varieties has been widely suggested . Even with our current understanding of varieties’ climate niches, only a few existing cultivars are late ripening enough to avoid the warming predicted to occur during maturation in future climate scenarios . We identified many late ripening varieties that can be tested in future studies for suitability in California . International projects such as ADVIDCLIM are currently testing phenological models of grapevine with the expectation that varieties planted will need to change in future climate conditions . Hypothetical crosses between very late ripening varieties were modelled and still struggle to be late-ripening enough to endure the predicted 23-day shift and increase of 7°C expected by the end of this century . Within existing varieties, clonal variation does not offer a wide enough plasticity for adapting to climate change, however, taking advantage of existing varieties in warm regions to grow as alternatives is a promising strategy .

Tissue differences were more revealing at the stage when flavors peak; 4865 transcripts that were significantly higher in skins compared to pulp at 23.2 °Brix were tested for over represented GO functional categories . Some of the top GO categories included photosynthesis, isoprenoid biosynthesis, and pigment biosynthesis . Some of the transcripts with the largest differences between skin and pulp at 23.2 °Brix are β-ketoacyl-CoA synthase , taxane 10-β-hydroxylase , wax synthase, a lipase, an ABC transporter, and phenylalanine ammonia-lyase . The abundance of 5716 transcripts was significantly higher in pulp than skin at 23.2 °Brix . Some of the top GO categories over represented were a variety of transport processes and small GTPase mediated signal transduction . Some of the transcripts with the largest differences in abundance with pulp greater than skin at 23.2 °Brix were polygalacturonase , flavonol synthase, stachyose synthase, an amino acid transporter, a potassium channel , and HRE2 . The transcript abundance of 2053 genes had significantly differential expression across °Brix levels and tissues . The top GO categories over represented in this set involved photosynthesis and phenylpropanoid metabolism, both associated with the berry skin .

Other flavorcentric categories of the 57 categories over represented include aromatic compound biosynthesis, 10 liter drainage pot fatty acid metabolism and alcohol catabolism. This transcript set was further analyzed by dividing into 10 clusters using k-means clustering . The over represented GO categories were determined for each cluster . Eight of the 10 clusters had distinct over represented GO categories; two clusters did not have any over represented GO categories, meaning that the genes in these two clusters were assigned to GO categories of expected proportions when compared to the entire NimbleGen array. Clusters 1, 8, 9 and 10 had a large number of over represented categories. Many GO categories within a cluster are subsets of others in that cluster and were grouped together. For example, cluster 4 had four over represented GO categories, oxygen transport, gas transport, heat acclimation and response to heat. The four categories could be grouped into two, as two are subsets of the others; this is how they were listed in Table 1.It would be impossible to discuss here all the transcript abundance changes detected in these berries. As we were interested in compounds associated with berry flavors as they develop or change in the late stages of berry ripening, we took a more targeted approach for analysis with this in mind.

Berries at 24° Brix are known to be near-optimal for flavor, thus we took a simple approach to look for genes that were peaking around this stage. We found some significant and large increases in transcript abundance between the 22.6 and 23.2 °Brix levels. A group of VviERF6 transcription factor paralogs represented 6of the top 10 transcripts increasing in transcript abundance from 22.6 to 23.2 °Brix in the skin, but not in the pulp . These VviERF6 TFs were also found in Cluster 8 . This is very interesting since many flavor compounds are derived from the skin and ERF TFs are known to be responsive to ethylene, a known fruit-ripening hormone. These VviERF TFs were named ERF105 in the annotation by Grimplet et al. , however they are more orthologous with AtERF6 as determined by a more comprehensive phylogenetic method using many plant species at Gramene . Annotation details of the V1 gene models of the VviAP2/ERF superfamily can be found in Additional file 8 including updated Vvi symbols according to its closest Arabidopsis ortholog as instructed by the Grapevine Gene Nomenclature System developed by the International Grape Genome Program Supernomenclature committee. This renaming of the AP2/ERF super family should facilitate comparative analyses and functions with other species, particularly Arabidopsis. To properly annotate the AP2/ERF super family of Vitis vinifera according to the IGGP Supernomenclature committee instructions, a phylogenetic tree was generated for the AP2/ERF super family of Arabidopsis thaliana and Vitis vinifera using the TAIR 10 and V1 gene models, respectively .

The labeled family classifications were derived from the Arabidopsis naming scheme by Nakano et al.. There are 130 members in the VitisAP2/ERF superfamily in the Pinot Noir reference genome. However, the six paralogs of ERF6 discussed above belong to a Vitis vinifera clade in subfamily IX and are distinctly different or separate from any Arabidopsis subfamily IX ERF TFs . All of these TFs in this clade are orthologs of AtERF6. VviERF6L1 [UniProt: F6I2N8; VIT_16s0013g00900] had one of the most interesting profiles of the 12 members of this clade because its transcript abundance peaked at 23.2 °Brix . Using k-means clustering, VviERF6L1 fell within Cluster 8 with 369 transcripts, including five additional VviERF6 paralogs. The top GO categories associated with Cluster 8 were genes associated with terpenoid metabolism and pigment biosynthesis . Other interesting flavor associated categories included fatty acid and alcohol metabolism . Representative transcripts from Cluster 8 that were correlated with the transcript abundance profile of VviERF6L1 can be seen in Figure 4. These are ACC oxidase, which is involved in ethylene biosynthesis; a lipoxygenase, part of a fatty acid degradation pathway giving rise to flavor alcohols such as hexenol; α-expansin 1, a cell wall loosening enzyme involved in fruit softening, and two terpene synthases, which produce important terpenes that contribute to Cabernet Sauvignon flavor and aroma. The high similarity of these transcript profiles indicates that ethylene biosynthesis and signaling may be involved in the production of grape aroma. Supporting this argument, two recent studies have shown that a tomato ERF TF , falling in the same ERF IX subfamily, has a strong effect on ethylene signaling and fruit ripening. The transcript abundance of AtERF6 in Arabidopsis is strongly increased by ethylene, which is triggered by the MKK9/MPK3/MPK6 pathway. The transcript abundance of VviMKK9 in the Cabernet Sauvignon berries was higher in the skin than the pulp, but there were no significant differences for VviMPK3 or VviMPK6 . This is not too surprising since AtMKK9 activates AtMPK3 and AtMPK6 by phosphorylation. In addition, the transcript abundance of AtERF6 in Arabidopsis increases with ROS, SA, cold, pathogens, and water deficit. There were no visible signs of pathogen infection in these berries. Additional circumstantial evidence for ethylene signaling in the late stages of berry ripening was that the transcript abundance of many VviERF TFs was significantly affected by berry ripening and/or tissue . The transcript abundance of 129 members from the berries was determined to be above background noise levels on the microarray . The expression profiles of the 92 significantly affected AP2/ERF superfamily members were separated into six distinct clusters by hierarchical clustering and indicated that this super family had a complex response during berry ripening . The 12 members of Cluster 1 responded similarly in both the skin and pulp, gradually decreasing with increasing °Brix with a large decrease in transcript abundance at the 36.7 °Brix level. Cluster 2 with 14 members, including 8 members of the VviERF6 clade, had much higher transcript abundance in the skin with a sharp peak at 23.2 °Brix. Cluster 3 had similar profiles in both the skin and pulp with a peak abundance at 25° Brix. Cluster 4 with 7 members was a near mirror image of cluster 2, 25 liter pot with a sharp valley for transcript abundance in the skin between 23 and 25 °Brix. Cluster 5 had 36 members with a steady increase in transcript abundance in the pulp but no substantial increase in the skin until 36.7 °Brix. Finally, in Cluster 6, there were 13 members with a higher transcript abundance in skins compared to pulp. Their transcript abundance increased with increasing °Brix level, but decreased in the skin. The transcript abundance of important components of the ethylene signaling pathway characterized in Arabidopsis and presumed to be functional in grape were also affected by °Brix level and tissue .

Three different ethylene receptors, VviETR1, VviETR2, and VviEIN4 decreased with °Brix level in the skin, however there was very little or no change in the pulp. Likewise, VviCTR1, another negative regulator of ethylene signaling that interacts with the ethylene receptors, decreased between 22.6 and 23.2 °Brix in both the skin and the pulp. The transcript abundance of the positive regulator, VviEIN2, peaked at 25 °Brix in both the skin and the pulp. AtEIN2 is negatively regulated by AtCTR1 and when it is released from repression, turns on AtEIN3 and the ethylene signaling pathway downstream. The transcript abundance of VviEIN3 increased with °Brix level, peaking at 25 °Brix in the skin, and was much higher than in the pulp. Although more subtle, its profile was very similar to VviERF6L1. Derepression of the negative regulators and the increase in positive regulators indicated that ethylene signaling was stimulated during this late stage of berry ripening.The transcript abundance of many of the genes involved in the isoprenoid biosynthesis pathway peaked between 23 and 25 °Brix level, particularly in the skin; this stimulation of transcript abundance continued in both the carotenoid and terpenoid biosynthesis pathways . DXP synthase is a key regulatory step in isoprenoid biosynthesis and its profile was similar to VviERF6L1; its transcript abundance was correlated with the transcript abundance of several terpene synthases in the terpenoid biosynthesis pathway . About 50% of the putative 69 functional terpene synthases in the Pinot Noir reference genome have been functionally characterized. Another 20 genes may be functional but need further functional validation or checking for sequencing and assembly errors. On the NimbleGen Grape Whole-Genome array there are 110 probe sets representing transcripts of functional, partial and psuedo terpene synthases in Pinot Noir . It is uncertain how many may be functional in Cabernet Sauvignon. There were 34 probe sets that significantly changed with °Brix or the °Brix and Tissue interaction effect; 20 of these are considered functional genes in Pinot Noir. Terpene synthases are separated into 4 subfamilies in the Pinot Noir reference genome; they use a variety of substrates and produce a variety of terpenes. Many of these enzymes produce more than one terpene. The top 8 transcripts that peaked in the skin at the 23.2 to 25 °Brix stages were also much higher in the skin relative to pulp . Five of the eight probesets match four functionally-classified genes in Pinot Noir ; these terpene synthases clustered very closely with VviTPS54, a functionally annotated – Linalool/- Nerolidol synthase. VviTPS58, a -geranyl linalool synthase, was also in the cluster. The other two probesets match partial terpene synthase sequences in the Pinot Noir reference genome. The transcript abundance of genes involved with carotenoid metabolism also changed at different °Brix levels and with tissue type . CCDs are carotenoid cleavage dioxgenases and are involved in norisoprenoid biosynthesis. The transcript abundance of VviCCD1 changed significantly with °Brix level and was higher in skin than pulp, except at 36.7 °Brix. Likewise, the transcript abundance of VviCCD4a and VviCCD4b changed significantly with °Brix level, but was higher in the pulp than the skin. The transcript abundance of VviCCD4c significantly increased with °Brix level, but there were no significant differences between tissues. VviCCD1 and VviCCD4 produce β- and α-ionone , geranylacetone , and 6-methyl-5-hepten-2-one in grapes. The transcript abundance of VviCCD8 significantly increased with°Brix level and was higher in pulp than skin. Phytoene synthase, which was also increased in the skin compared to the pulp , and VviCCD1, have been associated with β-ionone and β-damascenone biosynthesis. Other important grape flavors are derived from the fatty acid metabolism pathway and lead to the production of aromatic alcohols and esters. The transcript abundance of many genes associated with fatty acid biosynthesis and catabolism changed with °Brix level . In particular the transcript abundance of a number of genes were correlated with the transcript abundance of VviERF6L1 including VviACCase, Acetyl-CoA carboxylase; KAS III ; VviOAT, ; VviFAD8; ; VviLOX2 and VviHPL . The transcript abundance of alcohol dehydrogenases was affected by tissue and °Brix level . Some ADHs are associated with the production of hexenol and benzyl alcohol. Methoxypyrazines give herbaceous/bell pepper aromas. They are synthesized early in berry development and gradually diminish to very low levels at maturity. Nevertheless, humans can detect very low concentrations of these aroma compounds. Four enzymes, VviOMT1, VviOMT2, VviOMT3 and VviOMT4 , synthesize methoxypyrazines.

The Chern number is just a property of a band and does not come with an energy scale, so there is no reason to expect to encounter Chern bands only at low temperatures. Indeed, bands with finite Chern numbers have been shown to support quantized Hall effects in graphene quantum Hall devices at room temperature and high magnetic fields, as illustrated in Fig. 8.5A,B. The energy scale in a Chern magnet is set by the band gap produced by magnetic interactions. So if we’d like to know what the maximum temperature at which we can expect to find Chern magnets is, we need to think about the energy scales of known magnets. Magnetism is an interaction-driven electronic phase, and interaction-driven phases almost always melt at sufficiently high temperatures. However, among interaction-driven electronic phases ferromagnetism is particularly stable. Many common transition metals, including iron, cobalt, and nickel, support ferromagnetism into the range 600-1200 K, and all of these have found applications in a variety of electronic technologies as a result. These are of course all three dimensional crystals, vertical farm tower and Chern magnets are two dimensional crystals. So the next question we can ask is: do two dimensional magnets exist with Curie temperatures as high as room temperature?

The answer turns out to be yes, as illustrated in Fig. 8.5C,D. This magnetic system appears not to be a Chern magnet, unfortunately, but the point is that there is nothing in particular stopping a Chern magnet with a Curie temperature above 300 K from existing. The first intrinsic two dimensional ferromagnets were discovered in 2017, so I think it’s safe to say that our field hasn’t yet come particularly close to identifying all possible two dimensional magnets. It’s hard to do an accurate accounting of all of the so-far discovered two dimensional magnets, and it is certainly the case that many of these are are not Chern magnets. But of the two dimensional magnets we have found, a surprisingly large fraction are intrinsic Chern magnets. We know of eight intrinsic Chern magnets stable in the absence of an applied magnetic field in the published literature so far. These are presented, along with a few of their basic properties, in Table 8.1. We have discussed several of these materials in this thesis, but we have also skipped a few,including the only currently known intrinsic Chern magnet in an atomic crystal, i.e., not on a moir´e superlattice: MnBi2Te4. These other materials all also represent areas of active research. Of the Chern magnets we know about, 2/8 have C < 0 with B > 0, so that property might be quite common. Indeed, there’s no particular reason to expect the B > 0 ground state to have one sign of the Chern number over the other as far as I know. It’s worth mentioning that if we ever find one, a room temperature Chern magnet with C < 0 for B > 0 would also have extremely large ∆m, and will therefore likely be switchable, since ∆m increases linearly with EGap.

It is also the case that two of these materials have been observed to be switchable with pulses of electric current, although it is not yet clear if the tBLG/hBN and ABMoTe2/WSe2 Chern magnets share a common current-switching mechanism, or if their respective mechanisms would generalize well to large gap Chern magnets. I think it’s clear that we are in the early days of the study of this class of material systems, and without discovering more Chern magnets there is little we can say with much generality. All of this is to say that I don’t think it’s crazy to expect to discover Chern magnets at much higher energy scales than we have already encountered, and that should we ever find such a system, there are a variety of intriguing technological applications for which this class of material systems could be exploited.Over the course of my PhD, four nanoSQUID microscopes were proposed, and construction began in some form on all of them. By the time I left we had finished three of these microscopes. The first nanoSQUID microscope we completed was inserted into a bath of liquid helium and could operate at 4 K. The CrI3 magnetic imaging campaign was performed in this system. The second nanoSQUID microscope had a pumped He-4 evaporative cooling pot, and could reach temperatures of 1.5 K. The tBLG/hBN Chern magnet transport measurements, the tBLG/hBN Chern magnet imaging measurements, and the AB-MoTe2/WeSe2 Chern magnet imaging measurements were all performed in this system. The third nanoSQUID microscope had a closed cycle He-3 sorption pump cooling system, and could reach 300 mK. The fourth and final microscope remains under construction, and is designed to operate inside of a dilution refrigerator.

Pictures of several of these microscopes are shown in Fig. 8.6. Acoustic isolation chambers and the 300 mK system are not shown. All nanoSQUIDs have liquid He-4 baths for primary stage cooling, and all are mounted on several thousand pound vibration isolation tables floating on air legs to protect the nanoSQUID sensors from mechanical and acoustic shocks close to the surface. The nanoSQUID sensor circuit is fairly simple, with only one important non-standard circuitelement in it, other than the nanoSQUID itself of course. This is the series SQUID array amplifier. Current is forced into the nanoSQUID sensor in parallel with a shunt resistor of comparable resistance to the nanoSQUID sensor in the voltage state, which is generally a few Ohms. Current through the nanoSQUID side of the circuit is inductively coupled to a series of identical SQUIDs. These SQUIDs in series generate a large voltage, which is detected at room temperature. Current is forced through a feedback coil to maintain constant flux through the SQUIDs in series. This allows the circuit to maintain sensitivity over a wide range of currents . This current amplification circuit has good current sensitivity and enormous dynamics range, easily able to accommodate the several hundred µA necessary to reach the critical current of the nanoSQUID sensor. There are a lot of things that make scanning probe microscopy tough relative to other techniques for performing microscopy. One particularly challenging issue is navigation of the sensor to the sample. Those experienced with optical imaging might be spoiled by a contrast mechanism that is sensitive to a ton of different phenomena- the nanoSQUID can only see local gradients in magnetic field and temperature, vertical plant tower and those are rare unless you have intentionally built structures and devices that generate them for use in navigation. In particular, large thermal gradients and variations in local magnetic field aren’t general properties of surfaces, so it’s very easy to blunder a nanoSQUID sensor into a surface without ever seeing it coming! Experiments are thus much safer and more expedient if we can provide the nanoSQUID sensor with topographic feedback- i.e., some way of detecting surfaces without crashing into them and destroying the sensor. We did this using shear force microscopy, which is a form of atomic force microscopy, or AFM. There is nothing particularly atomic about this contrast mechanism in the nanoSQUID microscope- we don’t have nearly that much resolution- but it is incredibly useful for navigation because it allows us to safely and reliably detect surfaces without destroying the SQUID. Researchers and companies building scanning tunneling microscopes will often accomplish this by gluing their sensor, which is a microscopic metallic wire, onto a piezoelectric tuning fork and then exciting the tuning fork at its resonant frequency. This is a good strategy, but it must be modified for use with the nanoSQUID sensor, because the nanoSQUID sensor is considerably more massive than scanning tunneling microscope wires, so it cannot be glued onto the tuning fork without destroying its quality factor. We preserve the tuning fork’s quality factor by instead pressing a piezoelectric tuning fork against the side of the nanoSQUID sensor and performing shear force microscopy instead of tapping mode microscopy.

The glass micropipettes serving as substrates for the nanoSQUID sensors are so thin that they bend easily when pressed agains the tuning fork, and this keeps them in mechanical contact with the fork. An optical microscope image of a nanoSQUID sensor pressed against a tuning fork is shown in Fig. 8.8A, and the resonant frequency of the piezoelectrically driven tuning fork is shown in Fig. 8.8B, with a fit to a Butterworth Van-Dyke model. A phase-locked loop and PID feedback system together allow us to approach the surface with the nanoSQUID sensor, detect it without crashing into it and destroying the tip, and maintain feedback while scanning. Schematics of this assembly are shown in Fig. 8.9. A calibration of the scan range and height of the nanoSQUID AFM is shown in Fig. 8.10, with a comparison to a Bruker Icon AFM displayed as well.By far the most common experimental campaign for the nanoSQUID microscope during my time in Andrea’s lab involved being handed a sample fabricated primarily for transport or capacitance measurements, with little consideration afforded to the viability or ease of a scanning probe microscopy campaign on the sample. I think this is fairly common in scanning probe microscopy, and it often means that we need to get sensors to samples without much in the way of navigation infrastructure. For this reason the vast majority of nanoSQUID microscopy campaigns start with thermal navigation. Before cooling down the nanoSQUID microscope, an attempt is made to align the nanoSQUID sensor with the heterostructure under an optical microscope, but the nanoSQUID sensor often still starts several hundred microns away from the sample. Once the system is cold, we generally proceed by injecting a few mBar of helium gas into the sample chamber. This facilitates thermal transport between the nanoSQUID sensor and the sample. We then run an AC current through the sample, heating it and generating an AC temperature distribution. The nanoSQUID sensors are excellent thermometers as well as magnetometers, so we can use this thermal gradient to navigate to the sample. An image of the resulting distribution of temperature over the device is shown in Fig. 8.13A. Some of the details are described in a later section, but in summary this technique works surprisingly well- we can usually find samples even several millimeters away from the nanoSQUID sensor using this technique. Once the nanoSQUID is reasonably close to the sample, it is usually necessary to pump out the heat exchange gas before attempting magnetic imaging, since thermal contrast can produce large backgrounds. After the heat exchange gas is removed, further navigation must proceed by imaging the magnetic fields produced by applied current through the Biot-Savart effect, as illustrated in Fig. 8.13B. There are a few engineering challenges associated with fabricating nanoSQUID sensors. I will briefly describe a particularly challenging one in this section. Many of the best elemental superconductors are soft, heavy metals with low melting points like lead and indium. As any person who has spent some time in an experimental physics laboratory knows, solder doesn’t wet too many materials well, and it certainly doesn’t wet glass, so these metals tend to form droplets when deposited onto glass substrates. To form a uniform film, the superconducting metal must freeze instantly upon landing on the glass micropipette. To make sure this occurs, we must cryogencially cool the glass micropipettes while evaporating the superconducting metal onto them. This process involves specialized machinery that is covered in great depth in other documents and publications, so I won’t discuss it here. However, I do want to discuss the nature of the failure modes of this process. When liquids don’t wet surfaces well, they dewet into droplets, and these droplets tend to get more spherical and less film-like the worse they wet the surface. If this process is allowed to proceed to its conclusion before deposited metal solidifies, the resulting films won’t be connected at all, and your nanoSQUID circuit will be open. If the substrate is cold enough, the resulting film will at least be continuous, and it is likely that you will get a nanoSQUID. However, the formation of droplets is impossible to completely stop, especially near the edges of films and on the oblique surfaces of the nanoSQUID sensor . These droplets generally won’t short the sensor, but the nanoSQUID sensor is so small that electrons can reach these droplets through tunneling processes. Whenever droplets form between the two superconducting contacts on the nanoSQUID sensor electrons can tunnel between the contacts through the droplet, with the droplet functioning as a quantum dot.

This procedure will also produce strong magnetic signals if magnetism couples strongly to current in the two dimensional crystal we’re studying, and this will be the case in several of the systems we will discuss later. As I mentioned either, the nanoSQUID is well-thermalized to its environment, and its properties are quite sensitive to temperature, so we can use the nanoSQUID microscope as a sensitive nanoscale thermometer. I will not present any scientific conclusions based on scans performed using this technique, but it is a powerful capability and it is also useful more practically for nanoSQUID navigation, which is discuseed further in the appendix. Electron spin is a fundamentally quantum mechanical property; it can be more or less understood using analogies to classical physics, but it also has some properties that don’t have simple classical analogues. Spin can be understood as a quantized unit of angular momentum that an electron can never be rid of. Although an electron is, as far as we know, a point particle, this unit of angular momentum couples to charge and produces a quantized electron magnetic moment, which we call the Bohr magneton, µB. Electron spins both couple to and emit local magnetic fields, stackable flower pots and they are orthogonal to the electronic wave function- changing an electron’s wave function will not under normal circumstances influence its spin, and vice versa.

Electrons are fermions; they obey the Pauli exclusion principle, which states that no two electrons can be placed into the same quantum state. The simplest consequence the existence of electron spin has is the fact that electronic wave functions can fit two electrons instead of one, because an electron can have either an ‘up’ spin or a ‘down’ spin. We say that electron spin produces an energetic degeneracy, because each electronic wave function can thus support two electrons. Electron spin is not the only degree of freedom that can produce energetic degeneracies; we will discuss a different one later. All of the above arguments apply for electron spin in condensed matter systems as well, and we can expect every electronic band to support both spin ‘up’ and spin ‘down’ electrons. These arguments say nothing about interactions between electrons, and all of the physical laws we normally expect to encounter still apply. In particular, electrons of opposite spin can occupy the same wave function, but a pair of electrons have like charges, so they repel each other. There is thus an energetic cost to putting two electrons with opposite spin into the same wave function, and this cost can be quite large. This consideration is outside the realm of the physical models we have so far discussed, because electronic bands in the simplest possible picture are independent of the extent to which they are filled. We are introducing an effect that will violate this assumption; the energies of electronic bands may now change in response to the extent to which they are filled.

In particular, when an electronic wave function is completely filled with one spin species , it will remain possible to add additional electrons with opposite spins, but there will be an additional energetic cost to doing so. It is important to be precise about the fact that the displacement of the unfavorable spin species upward in energy occurs after the wave function is filled with its first spin. As a result, which spin species gets displaced upward in energy is arbitrary, and is determined by the spin polarization of the first electron we loaded into our wave function. This is an example of a ‘spontaneously broken symmetry,’ because before the addition of that first electron, the two spin species were energetically degenerate, and after the band is completely filled with both electron species, they will again be energetically degenerate. All of the above arguments apply to localized electronic wave functions and do not say anything specific about condensed matter systems, which involve many separate atoms that each support their own wave functions. A similar but somewhat subtler argument applies to electronic wave functions on adjacent atoms in condensed matter systems. When electronic wave functions on two adjacent atoms overlap, the structure of the delocalized electronic band that will emerge from them when they hybridize depends strongly on their relative spin polarization. When electrons on adjacent atoms have the same spin, the Pauli exclusion principle will prevent them from overlapping, thus minimizing their Coulomb interaction energy.

When electrons on adjacent atoms have opposite spins, the Pauli exclusion principle doesn’t apply, because the two electrons are already in different quantum states, and they can overlap. This produces a larger interaction energy for arrangements wherein electrons on adjacent atoms have antialigned spins . Like all qualitative rules there are exceptions wherein other energetic contributions are more important, but this argument applies to a wide variety of condensed matter systems. These systems are known as ‘ferromagnets.’ They have interaction-driven displacements of minority spin bands, are at least partially spin polarized, and have electron spins that are largely aligned with each other. Both of these energy scales, the ‘same-site interaction’ and the ‘exchange interaction’ respectively, can be quite large in real condensed matter systems. The presence of these effects can produce a variety of phenomena. The displacement of a spin subband upward in energy can produce partially spin-polarized metals , fully spin-polarized metals which we call ‘half-metals’ , and spin-polarized insulators which we call ‘magnetic insulators’ . Examples of each of these kinds of systems are known in nature, and all of these phenomena represent manifestations of magnetism. In principle one must perform calculations to determine whether magnetism will occur in any specific system. In practice there exist good rules of thumb for making qualitative predictions. Same-site interactions and exchange interactions minimize energy by minimizing the number of minority spin species present in a crystal, and putting the electrons that would otherwise have occupied minority spin states into majority spin states. Of course, this process always requires that the system pay an additional energetic cost in kinetic energy, because those previously unoccupied majority spin states started out above the Fermi level. The competition between these energy scales determines whether magnetism will occur in any particular material. It follows that systems with a multitude of quantum states with very similar energies in their band structure will be more likely to form magnets; to put it more precisely, we are looking for situations in which, near the Ferm level at least, E = C, where C is some constant. We can say that under these circumstances, the energies of electrons in the crystal are independent of their momenta. We can also say that we have encountered a large local maximum or even a singularity in the density of states. We sometimes call this the ‘flat-bottomed band condition,’ or just the ‘flat band condition’ , and it can be made quantitative in the form of the Stoner criterion. Magnetism is perhaps the simplest phenomenon that can be understood in this context, but it turns out that this argument applies very generally, tower garden and physicists expect to find a variety of interesting phenomena dependent on electron interactions whenever we encounter these situations. It is important to be specific about what we mean by a flat band here: we expect to encounter magnetism whenever an electronic band is locally flat- it is fine for the band to have very high bandwidth as long as it has a region with E ≈ C. These systems will tend to produce magnetic metals. When we encounter bands that are truly flat- i.e., they have both weak dispersion and small bandwidths- we are more likely to encounter magnetic insulators, as illustrated in Fig. 2.3. Most electrons in condensed matter systems are not moving at relativistic velocities. However, in the outermost valence shells of very large atoms , electrons can end up in such high angular momentum states that their velocities become relativistic.

We can thus expect electrons in bands formed from orbitals supported by heavy atoms to respond to local electric potential variations as if they provide a local magnetic field. This phenomenon is known as spin-orbit coupling, and it provides a mechanism through which the energy of an electron spin can couple to the electrostatic environment inside of an atomic lattice. Predicting the global minima in energy as a function of spin orientation is very challenging, but it is often true that a discrete set of minima exist, and of course they must obey the symmetries of the atomic lattice. For this reason in many magnetic materials there is a discrete set of magnetic ground states defined by axes along which the electron spin can point. It is very often the case that there exist two global minima in energy that are anti-parrallel along an axis of high symmetry; when this is the case, we say that the system is an Ising ferromagnet. The axis along which the ground state spin orientation points is called the ‘easy axis.’We are now ready to discuss a real magnetic system. Chromium iodide is a two dimensional magnetic insulator. Systems like chromium iodide have properties that are easy to understand in the context of the models we have so far discussed: strong on site interactions and exchange interactions produce full spin polarization, an interaction-driven band gap, and aligned magnetic moments within a single layer. As a result, these systems are electrical insulators. They support magnetic domain dynamics, and there is a temperature TC above which they cease to be magnetized , although they remain insulators far above that temperature. Extremely weak out-of-plane bonds produce highly anisotropic cleavage planes and make it relatively easy to prepare atomically thin crystals mechanically. As in other systems, this does not mean we will always be studying monolayers of the material. Bilayers, trilayers, four-layer crystals, and even thicker flakes can all have properties that differ significantly from those of a monolayer, often for reasons that we can understand, and CrI3 is no exception. Although it isn’t particularly relevant to the physics of magnetism, it’s worth mentioning that all of the chromium halides are highly unstable compounds, and decompose in a matter of seconds when exposed to air or moisture. These materials are difficult to study under normal circumstances, but two dimensional crystalline samples can be prepared inside of an inert-atmosphere glovebox. They can also be sandwiched, or ‘encapsulated,’ between other two dimensional crystals. Two dimensional crystals are so flat that this process produces an air- and water-proof barrier and protects the encapsulated crystal from degradation in atmosphere, facilitating easy measurements with tools like the nanoSQUID. The crystalline structure of CrI3, projected onto a two-dimensional crystal, is visible in Fig. 2.6A. Unlike graphene, CrI3 has two different kinds of atoms in its unit cell; the chromium atoms are responsible for the magnetic moments producing magnetism. CrI3 has fairly strong spin-orbitcoupling, and thus strong Ising anisotropy, with magnetic moments pointing out-of-plane . Most of the other chromium halides also support magnetic order, although the precise nature of each of their ground states differs somewhat. Both CrI3 and CrBr3 have ferromagnetic in-plane interactions and strong Ising anisotropy, but CrI3 has antiferromagnetic out-of-plane interactions, meaning that in the magnetic ground state of the crystal adjacent layers have their spins antialigned . Interestingly, CrCl3 also seems to have ferromagnetic in-plane interactions, but it is likely that it is not an Ising or easy-axis magnet, and instead has its spins pointed in the in-plane direction and thus free to rotate. It is evidently the case that although these systems are structurally very similar and all have strong spin-orbit coupling, their magnetic interactions and magnetocrystalline anisotropies vary wildly in response to modest differences in their electronic structure. As a result of all of the arguments discussed previously in this chapter, a CrI3 monolayer has finite magnetization even in the absence of an applied magnetic field, and its magnetic order experiences hysteresis in response to variations in the applied magnetic field, as illustrated in Fig. 2.6D . Antiferromagnetic interactions between adjacent layers in CrI3 mix in an interesting factor that can be easily understood: flakes with an even number of layers have no net magnetization in the absence of an applied magnetic field, but develop finite magnetization at higher magnetic fields as the applied magnetic field overwhelms interlayer interactions and realigns each layer in turn with the ambient magnetic field .