The journey lasts about eight hours, furrowing major migration checkpoints at the border

In the global market, shade-grown coffee is often advertised using particular imagery, which is meant to remind us of “conserved natural forests” or places with vast natural resources, rather than agricultural systems where both humans and non-humans play an important role in processes of commodity production. The result has been the construction of a coffee imaginary that paints shade-grown coffee plantations as lush gardens with tropical birds, jaguars, and foraging tapirs. Although an attractive and marketable vision of the coffee landscape, this imaginary is an inaccurate representation of the social reality of these spaces. At the core of this coffee imaginary is a conservation narrative2 that places responsibility of environmental degradation and species extinctions on exploitation by humans, and growing human populations . As a result, humans are excluded from places and the use of natural resources commonly used for social reproduction and the sustenance of human livelihoods. This conservation narrative incorporates some aspects of the “protected-area” discourse of conservation, emphasizing the maintenance of critical ecosystem functions and structures . Although this conservation narrative is based on strong scientific evidence, it is often presented under a framework of “crisis” , which potentially dehumanizes places and intensifies the human-nature dichotomy through the exclusion of the human experience . The impact of this conservation narrative on human communities has been explored in the context of exclusionary practices around protected areas, such as national parks, coral reefs, and fisheries . However, despite the prevalence of this conservation narrative in organic shade-grown coffee plantations, container growing raspberries no studies have explored its impact on plantation workers, and whether the embodiment of such narrative through potentially exclusionary practices further marginalizes vulnerable peoples.

This is particularly relevant for three reasons: first, seasonal workers in labor-intensive systems are one of the most vulnerable and marginalized actors within the coffee production chain . Research suggests that about 30% of the coffee currently consumed worldwide comes from large plantation systems , where seasonal workers face food and labor inequalities . Second, although literature on organic agriculture suggests that organic markets and price premiums from certifications can potentially support livelihoods in rural areas, farm workers continue to suffer inequalities and forced labor . In fact, the priorities of such ecolabeling initiatives focus primarily on certification criteria and privilege ecological goals while paying scarce attention to social processes and labor issues , and fail to question inequalities experienced by farm workers . And third, although shade grown coffee has been a critical system for conservation efforts and in some cases, supports peasant households , it is possible that the conservation narrative in organic shade-grown coffee plantations helps construct a coffee imaginary that misrepresents the human experience. This chapter examines the tensions that arise when conservation narratives meet the everyday-lived-experience of migrant farm workers in organic shade-grown coffee plantations in Soconusco, Mexico. I draw attention to the ways in which conservation narratives embodied in organic shade-grown coffee plantations have material and symbolic effects on farm workers’ everyday lived-experience, and argue that they contribute to farm workers’ vulnerability and marginalization. The relevance of this work lies in exposing the social intricacies of coffee production and biodiversity conservation within this labor intensive system as I demystify coffee production as a fair and just imaginary. First, I address the labor aspect of coffee plantations in the Soconusco region. Second, I discuss conservation narratives in the context of organic shade-grown coffee plantations, and the ways in which conservation narratives are embodied in organic shade-grown coffee plantations.

Finally, I discuss the implications of these conservation narratives on the everyday-lived-experience of migrant farm workers.In order to understand conservation narratives in coffee plantations and how they are perceived and contested by farm workers, I carried out ethnographic research in an organic shade-grown coffee plantation in the Soconusco region. The extension of this plantation is approximately 300ha and can be categorized as a mix between traditional and commercial polyculture, with hired wage labor, both permanent and temporary. The epistemological basis of my research is rooted in the interpretivist tradition of anthropology using a phenomenological approach, which emphasizes the importance of symbols and experiences as well as individual opinions, values and categories to understand societies . This methodological approach allowed me to capture meaningful experiences of farm workers, as well as to understand how farm workers perceive themselves in the plantation. During the harvest season between October 2015-January 2016, I picked coffee with 15 families of migrant farm workers. I also lived in their shacks and joined them in daily activities such as collecting edible wild plants, hunting, and preparing meals. My research consisted of participant observation and informal interviews that took place while picking coffee during the day, in the evenings over meals, and while performing other activities. Along with my interlocutors I experienced the physical struggles of picking coffee and, in return for their time, I contributed my portion of harvested coffee at the end of each day to their totals. The population of migrant farm workers with which I carried out my research were mestizo from Guatemala, although indigenous laborers also work in the plantation during the earliest part of the harvest season. I complemented my ethnographic research with interviews of three coffee plantation owners which allowed me to understand their engagement with conservation narratives, as well as their own struggles as coffee growers in a highly competitive market.

Farm owners are often blamed for the social conditions experienced in their farms, yet we should not assume that owners can automatically change this reality . Therefore, research about farm workers should also consider the experiences of the growers themselves. As suggested by Holmes “The fact that the perspectives of farm management are generally overlooked, inadvertently encourages the assumption that growers may be wealthy, selfish, or unconcerned” . This may reinforce a superficial understanding of the reality of farm workers, and therefore the fact that the complexity of their struggles and structural challenges are not often recognized. Due to my interest in analyzing how conservation narratives weave with the popular public perception of shade-grown coffee, I also visited coffee plantation lodges along the touristic Ruta del Café , where I collected written comments representing the public discourse surrounding shade grown coffee plantations. Plantations are highly specialized, large-scale agricultural operations, which are characterized by their intensive use of capital investments, as well as the exploitation of wage labor . Although plantations are primarily concerned with the production of agricultural products grown on land, in scale and method of operation plantations are more akin to a modern factory or industrialized agriculture than they are to a small-scale family farm . The plantation model of agriculture has affected the ecologies of place, including the interaction between humans, non-humans, and their environment, as it embodies both the control of nature and of people. The essence of plantations–with defined social stratification and a controlling character, full labor control, and the transmission of agricultural management instructions from top to bottom – continues to have a presence in agricultural production in Latin America. In its origins, plantation economies were entirely controlled by foreign capital, and labor would be primarily imported, but profits would be invested overseas. Knowledge and technology were also imported from abroad, often by sending the owner’s offspring to their country of origin to study, as part of the colonial emulation of the plantation economy . The primal organizational aspects of plantations slowly disappeared in most places in Latin America, blueberries in pots giving way to communal land and small holdings that followed land reforms and land grabs lead by displaced peasants. However, in some places plantations continue to shape the landscape and the lives of people that live in, of and around plantations. This is the case in the Soconusco region of Chiapas, Mexico. Coffee production in the Soconusco region– one of the most important coffee producing regions in Mexico– is particularly interesting, as it played a key historical role in incorporating the state into the global capitalist market . By the end of the XIX Century, factors such as the economic policies of the Mexican government , strong foreign capital and adequate ecological conditions, allowed the expansion of coffee plantations in the Soconusco region . In its expansion, the coffee plantation economy became not only a powerful mode of production, but also a way of life for both plantation owners and laborers , which remains an integral part of the cultural identity of Soconusco. The expansion of German plantations in this region was characterized by the adoption of a production system that did not fall too far from the hacienda system of the pre-revolutionary period . Coffee production in large volumes was done primarily by these large plantations, which had access to commercialization routes and the required capital investments . Today, the subsistence peasant economy coexists alongside the export-oriented economy of coffee plantations, generating a rich cultural, social and economic rural patchwork . Plantations in Soconusco maintain a social organization based on a centralized political structure and the employment of wage labor . At the center of this organization is the Patron, or owner of the plantation who often lives outside of the plantation; followed by an administrator; a sideman or mayordomo; a set of foremen or caporales in charge of crews of laborers; and the laborers themselves .

Although labor has been historically sourced from the highlands of Chiapas, most laborers today are currently seasonal migrants that come from Guatemala, primarily during the harvest season . The increasing migration of labor from Central and South America directly to the United States has generated a labor shortage in the region of Soconusco , which in turn has promoted alternative strategies of labor recruitment, including the provisioning of temporary visas and permits granted by the patrons of large operations, extended for up to five years of work . Such permits are only legally granted to individuals and not to entire families, meaning that only one or two family members are legally represented in the migratory destination . This situation only exacerbates the already vulnerable position of migrant laborers in coffee plantations, where living and working conditions are overwhelmingly unfair . Once migrating, the vulnerabilities that come with illegal status and high dependency from the contractors increase the stress experienced by farm workers . Additionally, the barriers to transnational mobility limits the ability of farm workers to claim better labor conditions and wages . Issues of migration, illegal status, and poor working conditions are accompanied by differences among farm workers in terms of their ethnicity, farming abilities, and their permanency in the farms, as has been shown for other agricultural systems that rely heavily on migrant labor . In coffee plantations, multiple ecological knowledges and imaginaries meet farm workers’ experience, which makes farm workers important actors for providing meaning to these spaces despite the fact they are often overlooked in the coffee production cycle. The harsh reality of laborers in coffee plantations contrasts with the coffee imaginary of shade-grown organic coffee and fails to show its reality, therefore obscuring the lives of people . Additionally, the social sustainability of organic agriculture can be widely questioned for the contradictions posed to laborers. On the one hand, organic agriculture provides a safer space to laborers in terms of limiting the exposure to pesticides , while on the other hand, it does not address structural inequalities, occupational injuries, and other health related concerns common in the community of farm laborers . In an ideal world, the benefits of organic shade-grown coffee should be perceived not only through the conservation of biodiversity, but also through improving social justice and human livelihoods. Yet, as Guthman argues, “the organic movement has fallen woefully short of addressing the social justice issues that are often assumed to be part and parcel of organic farming” .The Soconusco region is famous for its Ruta del Café, repeatedly advertised as a “magical destination on the coast of Chiapas”. Plantation-style lodges embedded in the tropical perennial forest overlook what is hard to not categorize as a dreamlike landscape for the avid nature lover: a mosaic of greens, the coffee within the dense natural forest, the sound of the river, and the singing of the birds.

A central aim in ecology is to understand how diverse factors at local and regional scales influence community assembly

Overall, the gene pool of cultivated fig analyzed possesses substantial genetic polymorphism and exhibits narrow differentiation. It is evident that fig accessions from Turkmenistan are somewhat genetically different from the rest of the Mediterranean and the Caucasus figs. A long history of domestication and cultivation with extensive dispersal of cultivars has often resulted in a great deal of confusion in the identification and classification of cultivars.Coffee, a highly demanded commodity supporting the livelihoods of more than 20 million families , has been traditionally cultivated under the shade of trees within tropical and subtropical biodiversity hotspots, making it relevant for conservation, food provisioning for rural families and the delivery of ecosystem services, meaning the processes and conditions provided by natural ecosystems with the potential to sustain and fulfill human welfare . Important ecosystem services in coffee plantations are pest control and pollination . However, coffee agroecosystems continue to experience a dramatic change in their management, characterized by the reduction of shade trees and increased use of chemical inputs . This intensification has had severe ecological implications, including the decline of ecosystem services ; and social consequences like food insecurity and seasonal hunger for farming families . Much research on the benefits of biodiversity in coffee agroecosystems – has pointed out that highly bio-diverse habitats have the potential to sustain a number of species and interactions that support ecosystem services . However, less is known about the specific ecological mechanisms supporting more biodiversity and higher abundance of natural enemies of coffee pests in complex agroecosystems . Previous studies have suggested that resource heterogeneity, large pots plastic meaning the variability of available resources through space and time and accessible refuges provided by surrounding vegetation could be important factors for arthropod communities . Yet, this is still an ongoing scientific exploration in coffee systems .

A second area of attention in coffee studies comprises the benefits that humans perceive from the planned and associated biodiversity in coffee agroecosystems. Most of these studies have highlighted the importance of bio-diverse coffee smallholdings in delivering food security and contribute to livelihoods of peasant households . However, research has suggested that established and temporary farm workers are a highly marginalized sector of the coffee production chain , which indicates that further attention needs to be put in terms of the overall lived experience– and specifically food-related experiences– of this sector. Ecological theory provides foundations for explaining higher abundance and diversity of natural enemies in complex habitats. Two complementary hypothesis have been developed and are useful in this sense: the first one suggests that different habitat types– meaning a variety of plants, strata, microhabitats, the spatial arrangement and temporal overlap of the plants –can support greater biodiversity of predators and reduce crop damage . This hypothesis considers that an increase in resources such as floral and extrafloral nectar and provisioning of shelter through improving non-crop vegetation, favors generalist and specialist predators and parasitoids through the delivery of alternative food . A second hypothesis suggests that niche complementarity, which occurs when higher diversity in the system allows for a grater range of functional traits to be represented, favors a more efficient use of resources and promotes diversity at higher trophic levels . Following these hypotheses communities of natural enemies could potentially thrive in coffee agroecosystems to the extent that these systems provide the necessary resources to survive when the main prey are not available. My dissertation departs from two socio-ecological understandings: 1) Resource heterogeneity– expressed in temporal variation of a particular resource, differences in quality and nutritional variation of resources, or differences in microhabitats – is considered an important factor influencing communities.

More complex habitats provide diversity of niches and ways to exploit resources in a particular environment , which in turn supports more biodiversity , influences the distribution and interactions of species and may favor niche partitioning and species coexistence in a given environment . 2) Ecological diversity exists along a gradient of social complexity, allowing the co-creation of spaces. The subsistence value of bio-diverse coffee systems draws upon the potential of growing and using a variety of resources other than coffee within the agroecosystem and adjacent plots . There is an increase interest and awareness that biodiversity conservation can have positive effects on food production and livelihoods . Such is the case of traditional coffee polycultures and indigenous agroforestry systems . Despite benefits of highly bio-diverse coffee plantations, coffee farmers frequently experience seasonal hunger and food shortages . Food related challenges are mostly reported from smallholdings; however a smaller but important sector of coffee production happens in large plantations, which represent a historically and currently important sector of the coffee production in Central America and issues of food security have not been explored enough. Under this framework, this dissertation evaluates three intertwined areas. First – I examine the influence of the availability of three resources on ant community dynamics such as colonization, reproduction, and two species interactions: predator-prey and parasitoid-host interactions. Ants and parasitoids are important natural enemies of coffee pests and are model systems to understand the mechanisms that favor diversity in complex habitats. Second, I examine the influence of local habitat factors on community dynamics. Third, I explore the every-day lived experiences of farm workers in coffee plantations, a scantily explored area that requires attention in coffee growing regions in Mexico. Community assembly is the process that leads to particular patterns of colonization of interacting species, that may share a particular resource , and a process that reflects survival of species in a particular habitat . The study of communities and their assemblage processes is important for explaining community dynamics, but also because it can provide important insights into spatiotemporal factors that maintain ecosystem services in face of global change, destruction of natural biomes, and intensification of managed systems .

Ants are a diverse and an interesting group of insects to use for studies of community assembly and drivers of coexistence because they are found almost everywhere and in the tropics they can represent up to 80% of the animal biomass . Understanding drivers of ant diversity and co-occurrence is of relevance, as ants participate in competitive, mutualistic and predatory interactions, as well as trait mediated interactions that often result in ecosystem services . Ants are important pollinators , predators of pests in agricultural systems , square planter pots seed dispersers and protectors of plants that provide resources useful for ants . Local and regional factors influence ant assemblages, however there is no single cause or dynamic that explains nest colonization patterns of entire communities of ants. Thus, recognizing that community assemblages can be structured through multiple ecological and evolutionary processes interacting synergistically is essential in community studies . By examining the community of arboreal ants that nest in hollow twigs in a coffee plantation, we investigated how availability of resources, such as diversity of nests with different sized entrances, and the vegetation strata in which nests are located influence colonization and nesting patterns for a community of twig-nesting ants. The role of cavity entrance diversity on Neotropical arboreal ants has been previously shown to strongly influence cavity colonization in a natural ecosystem . Although the present study shares a number of similarities with the previous study in terms of the experimental design, the novelty of our study lies in the examination of the assembly process of the arboreal ant community in an agroecosystem considering the vegetative strata as a potentially significant local factor influencing ant assembly. Other studies have also made important contributions to the understanding of the influence of resource availability, interspecific competition from dominant ants, and changes in environmental conditions on ant colonization, survival and community assembly ; similarly, studies have reported that niche differentiation and interspecific competition for similar resources structure ant communities . In the litter environment, factors such as patchiness in nest site availability can influence ground ants . For other communities, however, nesting sites might not be a limiting factor, although nest-site limitation may increase with agricultural habitat intensification or disturbance . Moreover, increases in diversity of nesting sites can influence species richness and composition . Only few studies examine factors that influence ant communities at the colonization stage, despite the importance of priority effects for community assembly . Recruitment limitation can affect colony density and incidence of less competitive species, thus examining initial phases of colonization may be important for understanding species coexistence . Moreover, the dispersal stage of colony formation maybe strongly influenced by community assembly mechanisms such as habitat filtering because ants must find suitable habitats .

The present study asked the following questions: 1) Does nest strata or diversity of nest entrance sizes influence the percent of nests colonized by arboreal twig-nesting ants, 2) Does nest strata or diversity of nest entrance sizes influence the species richness of arboreal twig-nesting ants colonizing nests? 3) Does nest strata or diversity of nest entrance sizes influence the community composition of twig-nesting ants colonizing nests? 4) Are nests with certain nest entrance sizes more frequently occupied, or have a higher species richness of ants? 5) Do individual ant species more frequently occupy nests in a certain strata or nests of a certain entrance size?We conducted field research in a 300-ha shaded coffee farm in the Soconusco region of Chiapas, Mexico between March and June 2012. The farm is located between 900-1100 m a.s.l. Between 2006-2011, annual rainfall at the farm was between 4000-5000 mm. During the time of the research, the production style of the farm could be classified as a mix of commercial polyculture and shaded monoculture according to the system of Moguel & Toledo . The farm has ~50 species of shade trees that provide 30-75% canopy cover to the coffee buses in the understory. We studied ant occupation of nests in 44 locations on the farm. Each study site was separated by a minimum of 100 m, and consisted of two neighboring Inga micheliana trees of approximately the same size and two coffee plants directly underneath the trees. In order to characterize the vegetation of each study site, we measured trees, canopy cover, and coffee density. For all measurements, we used the midway point between the two Inga micheliana trees as the center point. In a 25 m radius circle around the center, we identified and counted each tree and measured the circumference and height of all tress. We sampled canopy cover at the circle center, and 10 m to the N, S, E, and W of the circle center with a convex spherical densitometer. We counted the number of coffee plants within 5 m of each focal Inga tree in each site. With the vegetation data, we calculated a vegetation complexity index . To calculate the index, we divided values for each vegetation variable by the highest observed value for each variable. For the number of coffee plants and the percent of trees in the genus Inga, we subtracted the product from 1 as these two factors generally negatively correlate with vegetation complexity. Then, we took the average of all values for each site to obtain a single value between 0 and 1 .Treatments were randomly assigned to plants in each site. We attached nests to plants with twist ties and plastic string between 0.5 to 1.5 m above ground on coffee plants, and between 4 to 6 m above ground for Inga trees. We placed nests flush with coffee or tree branches. We placed nests between 5-7 March and harvested all nests 14 weeks later . The period of the study encompassed part of the rainy season. Rain and moisture have a significant effect on colony phenology because they regulate alate’s flights in the absence of temperature variation . Although nests were placed long enough to be colonized by ants, longer time periods may have allowed us to capture colonization dynamics across time. To determine effects of nest entrance size, entrance size diversity, and nest vegetation strata on colonization, we collected artificial nests, placed them in bags, froze them, and then cut open all nests to remove the contents. We noted whether each nest was occupied or not. We stored ants in 70% ethanol and later identified them according to the Ants of Costa Rica , and AntWeb . For all species found, we obtained an approximate head width measurement from AntWeb .

The experiment was arranged in a randomized complete block with four replications

Despite higher recorded GDD in the present study, titratable acidity at harvest was maintained around 7 g•L-1 . Ultimately the reduction in cluster temperature imparted by the shading impeded organic acid degradation therefore maintaining berry acidity. It was identified that anthocyanin accumulation was maximized at 875 GDD and a daily mean light intensity of 220klm⸱m-2 after which anthocyanin content decreased in Cabernet Sauvignon. Previous works that used partial shading that transmitted 60% of solar radiation had also resulted in increased anthocyanin content compared to unshaded fruit in under similar growing season and climatic conditions . In 2021, shade films did not affect the anthocyanin content in berry skins at harvest, due to the cooler growing season limiting anthocyanin degradation post-veraison. The reduction in anthocyanin content observed in 2020 may result from repressed anthocyanin biosynthesis at hot temperatures via the MYB4 repressor . However, it is also highly likely that elevated temperatures in 2020 resulted in increased anthocyanin degradation in exposed fruit compared to shaded fruit, leading to shaded fruit having greater anthocyanin content. Flavonols are photoprotectants and free radical scavengers in the plant kingdom . As such, these compounds are directly responsive to light exposure of the cluster. In the phenylpropanoid pathway, MYBF1 is a transcriptional regulator ofFLS, the key gene in flavonoid synthesis . It has been shown that MYBF1 is upregulated by UV-B light, resulting in increased flavonols in grape berry skins . Thresholds for optimal sunlight exposure have been elucidated in previous solar radiation exclusion experiments, where Martínez-Lüsher et al. tracked flavonol development over the growing season under 20% and 40% shading conditions. It was determined that net flavonol biosynthesis occurs until approximately 570 MJ m-2 of accumulated global radiation which corresponds with 7.6% molar abundance of kaempferol in grape skins .

Beyond these thresholds, plastic gutter flavonols started to be degraded in the grape berries. Our study showed a similar trend for flavonol content in hot years like 2020. The control treatments in 2020 exceeded 8.6% kaempferol abundance, while shade films were effective in maintaining kaempferol abundance below this overexposure threshold. In cooler years like 2021, flavonol degradation was not observed at the global radiation threshold as a result of the cooler growing season. Rather, biosynthesis continued to increase flavonol content until harvest in 2021. Shade films effectively lengthened the period of flavonol biosynthesis and reduced the amount of time during ripening where clusters are under flavonol degrading conditions. Anthocyanins are comprised of two aromatic rings linked by three carbons in an oxygenated heterocycle . Hydroxylation and methylation of the B- ring is responsible for color and hue of each anthocyanin molecule. Increasing free hydroxyl groups on the B-ring enhances blueness while methylation of the hydroxyl groups increases redder hues in grape skins . From a winemaking perspective, 3’4’5-OH anthocyanins are more resistant to degradation during fermentation, leading to stable wine color . In this study, overhead shade films did not affect anthocyanin hydroxylation by harvest in either year of this study. However, shifts in anthocyanin hydroxylation have been previously documented: colored shade nets reducing solar radiation by 40%, showed higher anthocyanin and flavonol hydroxylation compared to unshaded treatments . Previous studies reported increases in the ratio of di-tri hydroxylated anthocyanins in grapevines under water deficits . The absence of this shift in anthocyanin hydroxylation under shade films was most likely due to similar grapevine water status among the shaded and control treatments, as the vines were not under water deficit conditions. However, shade films altered flavonol hydroxylation under hot growing conditions in 2020, with hydroxylation being the highest in the least exposed shade films . Shade films D4 and D5 transmitted 60% and 40% of UV-B radiation respectively, resulting in less flavonol hydroxylation than D1 and D3, but more hydroxylation than the control. In cooler growing conditions in 2021, all shade films had comparable levels of flavonol hydroxylation, yet hydroxylation was still greater under shade films than the control.

These results may be due to the upregulation of flavonoid 3’ hydroxylase . This enzyme is responsive under sun exposure and is responsible for the generation of 3’4’ hydroxylated flavonoid precursors .It has been long recognized that the quality of wines is closely associated with the accumulation of secondary metabolites, specifically flavonoids and volatile organic compounds that have a direct effect on wine color, taste and aroma . Flavonoids in wine include anthocyanins, flavonols and flavan-3-ols. Wine color, particularly its hue and intensity, are strongly determined by anthocyanin methylation, acetylation, hydroxylation of the anthocyanin B-ring, and co-pigmentation with cofactors such as flavonols . Partial solar radiation exclusion was shown to effect anthocyanin hydroxylation. Tarara et al. demonstrated increased dihydroxylation of anthocyanins in grape berries exposed to direct solar radiation compared to shaded fruit. Likewise, Martínez-Lüscher et al. monitored anthocyanin hydroxylation under colored photo selective shade nets and found that by reducing solar radiation by 40% with black polyethylene shade nets, the ratio of tri- to dihydroxylated anthocyanins was increased compared to uncovered control fruit. Such shifts in anthocyanin hydroxylation can impact anthocyanin hue and wine antioxidant capacity. Wine aroma in both red and white wines is a matrix formed by a variety of volatile compounds. However, the composition of the matrix can be impacted by grape cultivar, vineyard conditions and fermentation conditions. Contribution of volatiles to wine flavor composition is related to its chemical structure . The most abundant class of volatile compounds found in the wine matrix are higher alcohols . These by-products of yeast nitrogen metabolism are usually described by unpleasant “solvent” or “fusel” aromas when present in concentrations greater than 400 mg/L . The more pleasant “fruity” aromas described in wine are associated with esters. Esters are often in highest concentrations in young red wines and decrease in concentration with aging . C-13 norisoprenoids and terpenes are key aromas compounds found in both red and white wines, contributing fruity and floral aromas at low olfactory concentrations . C-13 norisopenoids are understood to be derivatives of enzymatic or photochemical degradation of carotenoid pigments in the grape berry . In plants, carotenoids have photo protectant and antioxidant properties, making these pigments responsive to solar radiation in grape berries. Carotenoids in grape berries have been shown to increase in berries with increased in solar radiation pre-veraison . However, under extreme exposure to heat and solar radiation, there is a documented decrease in carotenoid concentrations during ripening . To preserve the carotenoid concentrations in the grape berry and to promote C-13 norisoprenoids in resulting wines under more frequent heat wave events and increases in air temperature, artificial shading with black polyethylene cloth has been trialed and found that shaded fruit contained more carotenoids than unshaded fruit . However, the effect of partial solar radiation exclusion on wine C-13 norisoprenoid content seems to be more nuanced. Wines produced from the shaded fruit contained more β- damascenone as well as esters compared to wines produced from unshaded fruit . Yet, there are conflicting reports showing no effect of UV exposure on β- damascenone concentrations in Shiraz wines made from clusters that underwent solar radiation exposure via varying rates of leaf removal and polycarbonate UV screens . Like C-13 norisoprenoids, final terpene concentrations in wines depends on the net accumulation in grape clusters exposed to excessive temperatures and UV radiation . The effect of photo selective overhead shade films on whole plant physiology and temporal development of berry flavonoids of Cabernet Sauvignon development over two growing seasons was previously studied in a hot region . Grape berries growing under reduced near-infrared radiation exposure in hotter than average years, resulted in a 27% increase in anthocyanin content at harvest than the exposed control due to decreases inanthocyanin degradation due to high berry temperatures . Moreover, flavonol degradation was similarly decreased, thus optimizing flavonol content in the grape berry under reduced near-infrared radiation exposure .

The objectives of this study aimed to determine the extent to which the impact of photo selective overhead shade films on flavonoid development transfer to wine and the cascading effects of partial solar radiation exclusion had on aroma composition of resultant wines. The experiment was conducted in Oakville, CA, USA during two consecutive growing seasons at the University of California Davis, Oakville Experimental Vineyard. The vineyard was planted with “Cabernet Sauvignon” clone FPS08 grafted onto 110 Richter rootstock. The grapevines were planted at 2.0 m × 2.4m and oriented NW to SE. The grapevines were trained to bilateral cordons, blueberry container vertically shoot positioned, and pruned to 30-single bud spurs. Irrigation was applied uniformly from fruit set to harvest at 25% evapotranspiration as described elsewhere . The photo selective shade film treatments were previously described in Marigliano et al. and their properties presented in Figure 1. Shade films were designed to target portions of the electromagnetic spectrum previously observed and measured at the experimental site . Briefly, four photo selective shade films and an untreated control were installed in 3 adjacent rows on 12 September 2019. The shade films remained suspended over the vineyard until 20 October 2021. The shade films were 2 m wide and 11m long and were secured on trellising approximately 2.5 m above the vineyard floor. Each experimental unit consisted of 15 grapevines in 3 adjacent rows. Grape clusters were harvested by hand from each experimental unit when berry total soluble solids reached 25o Brix on 9 September 2020, and 7 September 2021, respectively. Vinification was conducted in 2020 and 2021 at the UC Davis Teaching and Research Winery. Upon arrival at the winery, grapes were destemmed and crushed mechanically. Must from each field experimental unit was divided into three technical fermentation replicates . K2S2O2 was added to each treatment- replicate and must was allowed to cold-soak overnight at 5o C in jacketed stainless-steel tanks controlled by an integrated fermentation control system . The following day each treatment-replicate was inoculated with EC- 1118 yeast to initiate fermentation. Musts were fermented at 25°C and two volumes of must were pumped over twice per day by the integrated fermentation control system. During the winemaking process, TSS was monitored daily using a densitometer and fermentations were considered complete once residual sugar contents were less than 3 g⸱L-1 . Wines were then mechanically pressed using a screw-type basket press. Following pressing, wine samples were collected for flavonoid analysis. Malolactic fermentation was initiated with the addition of Viniflora® Oenococcus oeni . Malolactic fermentation was carried out at 20o C. Upon completion of MLF, free SO2 levels were then adjusted to 35 mg⸱L-1 and wines were bottled. Using a spectrophotometer , color intensity , hue, total polyphenolic index and % of polymeric anthocyanins was determined following procedures described by Ribéreau-Gayon, Glories, Maujean, and Dubourdieu . Wine samples were diluted in water and absorbance readings were taken at 280, 420, 520, and 620nm. The absorbance at 740 nm was subtracted from all absorbance readings to eliminate turbidity. CI was calculated as the sum of absorbance at 420, 520 and 620nm. Hue was calculated as the ratio between the absorbance at 420 and 520nm. The percentage of polymeric anthocyanins was determined via absorbance measurements at 520nm after anthocyanin bleaching with a sodium bisulfite solution . TPI was determined by diluting wines with water and recording absorbance at 280nm. Volatile compounds in wine samples were analyzed following procedures described previously . Briefly, 10-mL of each wine sample was transferred to a 20-mL amber glass vial . Each vial also contained 3 g of NaCl and 50μg of an internal standard solution of 2-undecanone . After agitating at 500 rpm for 5 mins at 30o C, samples were exposed to 1 cm polydimethylsiloxane/divinylbenzene/Carboxen , 23-gauge SPME fiber for 45 mins. Helium was used as a carrier gas at a flow rate of 0.8636 mL/min in a DB-Wax 231 ETR capillary column  with constant pressure and temperature at 5.5311 psi and 40o C, respectively. The oven temperature was kept at 40o C for 5 mins and then incrementally increased by 3o C/min until reaching 180o C. Oven temperature was then increased by 30o C/min until reaching 260o C, at which temperature was maintained for 7.67min. The SPME fiber was desorbed split mode with a 10:1 split for wine samples and held in the inlet for 10min to prevent carryover effects. The method was retention time-locked to the 2-undecanone internal standard.

The primary assembly is more complete and consists of longer phased blocks

DCA-treated colon cancer cells exhibited higher ROS generation, membrane blebbing, activation of mitochondrial apoptotic pathway, and formation of apoptotic bodies compared to untreated cells. DCA exposure also resulted in micronuclei formation accompanied by a dose-dependent response in NFκb activation that was attenuated by cotreatment. The above studies clearly demonstrate that hydrophobic BAs induce cellular oxidative stress through elevated reactive oxygen and nitrogen species production with similar results observed between human liver and colon cell lines. Interestingly, human HCC cells treated with hydrophobic primary CDCA also displayed cell cycle arrest, caspase-9-like activity, poly ADP-ribose polymerase cleavage, dose-dependent sites of DNA lesions, and extensive nuclear fragmentation. These cells also showed activation of ERK1/2 leading to the phosphorylation and stabilization of myeloid cell leukemia sequence 1 in a mitogenactivated protein kinase-dependent manner which conferred greater resistance to chemotherapeutic drugs. These findings strongly support that excessive levels of hydrophobic BA can exert a carcinogenic effect on enterohepatic tissues by promoting genomic instability through oxidative injury. Figure 3 provides an overview of the implicated carcinogenic consequences of BA dysregulation, gut dysbiosis, and insulin resistance. Several synthetic drugs such as colesevelam and colestimide, designed to sequester BAs, have demonstrated efficacy in improving insulin resistance and glucose tolerance. BA receptor agonists also appeared to be promising in the management of obesity and diabetes related symptoms. Specifically, synthetic retinoid such as acyclic retinoid was investigated for its beneficial effect on obesity-related liver tumorigenesis, since RXRα, a heterodimer partner of FXR, square plastic plant pot was found to be repressed in early stages of HCC due to phosphorylation by the Ras/MAPK signaling pathway. Results indicate that ACR inhibited the Ras/MAPK pathway, ameliorated liver steatosis, improved insulin sensitivity, and decreased inflammation.

Ultimately, ACR appears promising in restoring RXRα function in the liver, making it an effective chemoprevention drug against HCC progression. With the intestinal microbiota implicated as one of the key players in the progression of liver and colon carcinogenesis in obese and diabetic patients, selective modification of the gut microbial composition has been extensively researched as a viable alternative or additive to current treatment plans. Prebiotics promote the growth of beneficial bacteria while probiotics are live microorganisms, administered exogenously, which provide a benefit beyond nutrition. Most prebiotics exist in the form of non-digestible carbohydrates and exert protective effects against liver and colon cancer. Dietary fibers shortened GI transit time to reduce the length of exposure to toxic metabolites such as hydrophobic BAs and bacterial toxins while aiding in their incorporation in feces for excretion. In addition, these fibers enhanced the growth of beneficial bacteria such as Bifidobacteria, lowered intestinal pH, and inhibited the growth of harmful bacteria. An increase in bifidobacteria is the signature of prebiotic treatment using insulintype fructans or galactooligosaccharides. Other resistant dietary fibers such as pomegranate peel extract and natural phytoalexin resveratrol can alter the gut microbiome in favor of bifidobacteria while lowering inflammatory markers in the colon and visceral adipose tissue. Short chain fatty acids , the fermented end products of dietary fibers, are recognized for their ability to inhibit growth and promote apoptosis in colon and liver cancer cells. SCFAs can also activate various drug metabolizing enzymes to decrease DNA mutation and reduce cancer risk. Complementing SCFAs with fish oil selectively reduced unsaturated fatty acid accumulation in the liver, improving hepatic fat oxidation and inflammation. Furthermore, probiotics have been suggested as a tool to manage inflammatory bowel disease. Although no general consensus exists for the beneficial effects of probiotics in obese, diabetic, and NAFLD patients, reduced hepatic total fatty acid content and serum alanine aminotransferase levels were noted in rodent models treated with probiotics.

A synergistic effect of probiotics and blueberry husks in preventing colon carcinomas and subsequent liver tumors has been observed in rats. Blueberry husks alone significantly decrease the number of colonic ulcers and dysplastic lesions while probiotics alone improved liver function by decreasing parenchymal infiltration and bacterial translocation. Thus, the combination treatment of probiotics and blueberry husks delayed colonic carcinogenesis and prevented hepatic injuries. The combined treatment also reduced endotoxin-producing enterobacteriaceae and increased beneficial, anti-inflammatory lactobacilli. Synbiotics, mixtures of probiotics and prebiotics, were noted to lower colon cancer risk through improved insulin resistance, reduced inflammation, and preservation of gut barrier integrity in rats fed a high-fat diet. In terms of gut microbial ecology, synbiotic treatment significantly increased fecal Lactobacillus species at the expense of potentially pathogenic E. coli and Staphylococcus, thus lowering the endotoxin level in cirrhotic patients. Simple dietary changes can also aid in reversing BA dysregulation and intestinal microbiome derangement. Vitamin B6 can improve colon health by significantly reducing hydrophobic LCA levels in the colon, creating a reduced LCA to DCA ratio. LCA was 20- fold more toxic than DCA toward liver and colon cancer cells, and vitamin B6 helped with detoxifying enzymes and decreased DNA damage.170 Moreover, various naturally derived products are also under investigation including the antioxidant tempol which reduced obesity and improved insulin resistance in mice fed a high-fat diet by activating bile salt hydrolase to increase intestinal tauro-beta-muricholic acid, an FXR antagonist. Tempol appears to exert its antiobesity effects through FXR since inhibition of intestinal FXR promoted diet-induced obesity and insulin resistance. Tempol was also able to shift the gut microbial profile from firmicutes toward bacteroidetes dominance. Plant-based products such as burdock powder and genistein also showed moderate efficacy in normalizing BA homeostasis and the gut microbiome in animal models. The proposed beneficial effects on BA dysregulation and gut dysbiosis by pharmacological and dietary intervention are described in Figure 4. In this genome release, we report on the first assembled genome of a member of the genus Arctostaphylos. 

Our genome assembly is part of the California Conservation Genomics Project , the goal of which is to establish patterns of genomic diversity across the state of California and its many habitats. The CCGP will sequence the complete genomes of approximately 150 carefully selected species projects. Many of these taxa are threatened or endangered, and therefore in need of conservation management in the face of rapidly accelerating biodiversity decline. The combined reference genome plus landscape genomics approach of the CCGP, based on the resequencing of many individuals of each target species across the state, will allow the identification of hotspots of diversity across California and provide a framework for informed conservation decisions and management plans. Manzanitas are among the most conspicuous and dominant native chaparral species in the California Floristic Province , a biodiversity hotspot characterized by a Mediterranean-type climate with hot, dry summers and cool, wet winters. These plants comprise the most diverse woody genus in the CFP , and their diversity has long fascinated taxonomists. Manzanitas serve essential roles in their native ecosystems, including rapidly regenerating in fired-disturbed areas, and providing food resources for pollinators and fruit-eating animals . In addition, these plants are of great importance for conservation management: over half of the more than 100 morphologically defined manzanita species and subspecies are narrow endemics with highly restricted distributions and are considered rare and/or endangered . In contrast to their importance in ecology, evolution, and conservation studies, genomic resources for manzanitas are nearly nonexistent beyond investigations into karyotypes of diploid and tetraploid species . In this study, we present the first genome sequence of a manzanita. Big berry manzanita, Arctostaphylos glauca , is a widespread diploid species common in northern Baja California and across southern and coastal central California that is hypothesized to be the progenitor of several potential hybrid manzanita species . With funding and support from the CCGP, 25 liter square pot we created this scaffold-level assembly using a hybrid de novo assembly approach that combines Hi-C chromatin-proximity and PacBio HiFi long-read sequencing data. This genome assembly will provide a robust basis for studying the diversification and evolutionary history of Arctostaphylos in the CFP.A Dovetail Hi-C library was prepared in a similar manner as previously described . For each library, chromatin was fixed in place with formaldehyde in the nucleus. Extracted, fixed chromatin was digested with DpnII, the 5′ overhangs were filled in with biotinylated nucleotides, and free blunt ends were ligated. After ligation, crosslinks were reversed, and the DNA purified from protein. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to ~350 bp mean fragment size and sequencing libraries were generated using NEBNext Ultra enzymes and Illumina-compatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were prepared and sequenced on an Illumina HiSeq X by Dovetail Genomics .The CCGP assembly protocol version 1.0 uses PacBio HiFi reads and Hi-C chromatin capture data for the generation of high-quality and highly contiguous nuclear genome assemblies. The output corresponding to a diploid assembly consists of two pseudo haplotypes .

The alternate consists of haplotigs in heterozygous regions and is not as complete and more fragmented. Given the characteristics of the latter, it cannot be considered on its own but as a complement of the primary assembly . Arctostaphylos is the third genus with a genome assembly in the heath family, Ericaceae, following release of assembled genome sequences of Rhododendron and Vaccinium . These two genera are of significant economic importance: many species, hybrids, and cultivars of Rhododendron, including rhododendron and azalea, are important landscape and ornamental plants, and the fruits of many Vaccinium species, which include cranberry, blueberry, and huckleberry, are consumed by humans and other animals. Species in the Ericaceae are also notable for their ability to tolerate acidic and nutrient-poor soils that often characterize boreal forests and bogs, allowing them to thrive in habitats that are inaccessible to most species. Their tolerance for these conditions is due in part to the formation of mutualistic associations between the roots of the plants and soil fungi of a type unique to the heath family known as ericoid mycorrhizae. Ericoid mycorrhizae are distinct from common mycorrhizal associations found in most angiosperms, and are far less well understood . Complete genome sequences from three genera in this family will provide a strong foundation for investigating the basis of this unique mutualism and its ability to promote survival in inhospitable soils. The size of the A. glauca assembly is 547Mb, which is similar to the two Rhododendron genomes and half that of V corymbosum, which is tetraploid . The tetraploid nature of V. corymbosum also explains the vastly greater number of duplicated genes in its assembly compared to the two diploid assemblies. The scaffold N50 of the A. glauca assembly is longer than R. williamsianum, and close to R. simsii and V. corymbosum, suggesting that the contiguity of Arctostaphylos is comparable to the other taxa . Analysis using Repeat Modeler indicated that 57.71% of the A. glauca genome is composed of different categories of repetitive elements . In contrast, analysis using RepeatModeler identified only 26%, 47.5%, and 44.3% of the genome comprising repeat elements in R. williamsianum, R. simsii, and V. corymbosum respectively. The BUSCO completeness assessment of the A. glauca assembly is higher than R. williamsianum and close to the V. corymbosum , indicating that our final assembly is high quality . Overall, the A. glauca, R. simsii and V. corymbosum genomes are of comparably high contiguity and completeness. The lower contiguity and completeness of the R. williamsianum genome may be due to the lack of HiFi or other longread data in the assembly. This explanation is consistent with other studies demonstrating improved assembly with the inclusion of longer reads . Although the big berry manzanita is a common and widespread species, nearly half of the 60+ manzanita species are rare or threatened. Many are now represented by only one or two populations, and are thus vulnerable to complete eradication by the increasingly common and intense wildfires experienced across California each year. Our manzanita genome sequence will help fulfill the overall goal of the CCGP, serving as a key resource to assess genetic diversity in these threatened endemics and move forward with coordinated conservation programs.Sangiovese is the most important Italian red grape variety, with origins in Tuscany and Calabria in the south of Italy. Through time, Sangiovese has always been considered as a good-quality cultivar for wine production. Today, it is the basis of internationally known DOC and DOCG wines from Tuscany in Italy, the region where it is most cultivated. Sangiovese is also grown in Argentina, California, France, and in other countries such Australia and Chile, but to a lesser extent.

The remaining 3946 expressed clusters were either shared among groups of libraries or specific to unique libraries

In general, when the berries were green, the numbers of sRNA-generating loci located in genic and intergenic regions were roughly equal in all environments and for both cultivars, except for Sangiovese berries collected in Riccione, which show a slight intergenic disposition of sRNA-producing regions . Differently, in ripened berries on average 65% of the sRNA-generating loci were in genic regions, indicating a strong genic disposition of the sRNA-producing clusters . The shift of sRNA-producing clusters from intergenic to mostly genic is more pronounced in Cabernet Sauvignon berries collected in Riccione, with an increase of approximately 20% of expressed clusters in genic regions when berries pass from the green to the ripened stage. When comparing the clusters abundance among libraries, we found that 462 clusters were expressed in all libraries. Interestingly, 1335 of the 4408 expressed clusters were specific to Riccione-derived libraries . The other two environments showed a much lower percentage of specific clusters, 263 and 140 in Bolgheri and Montalcino respectively . Comparing the expressed clusters between cultivars or developmental stages, we did not observe a similar discrepancy of specific clusters toward one cultivar or developmental stage; roughly the same proportion of specific clusters was found for each cultivar and for each developmental stage . Among the 1335 specific clusters of Riccione, 605 were specific to Cabernet Sauvignon ripened berries of and 499 to Sangiovese green berries. Other smaller groups of expressed clusters were identified as specific to one cultivar, black plastic plant pots one developmental stage or also one cultivar in a specific developmental stage. When comparing the expressed clusters with the presence of transposable elements annotated in the grapevine genome , we noticed that approximately 23% of the sRNA-generating regions were TE-associated.

Sangiovese green berries from Riccione have the highest proportion of TE-associated expressed clusters, while Cabernet Sauvignon ripened berries also from Riccione show the lowest proportion of TE associated expressed clusters. Sangiovese berries have the highest percentage of expressed clusters located in TE when cultivated in Riccione, compared to the other two vineyards. Interestingly, Cabernet Sauvignon berries show the lowest proportion of TE-associated clusters when growing in Riccione , independently from the berry stage. In all the libraries, Long Terminal Repeat retrotransposons were the most represented TE. More specifically, the gypsy family was the LTR class associated with the highest number of sRNA hotspots. The other classes of TE associated with the sRNA-generating regions can be visualized in Figure 3B. To determine the global relationship of small RNA-producing loci in the different environments, cultivars and developmental stages, we performed a hierarchical clustering analysis. As showed in Figure 4, the libraries clearly clustered according to the developmental stage and cultivar and not according to the environments. Ripened and green berries had their profile of sRNA-generating loci clearly distinguished from each other. Inside each branch of green and ripened samples, Cabernet Sauvignon and Sangiovese were also well separated, indicating that, the cultivar and the stage of development in which the berries were sampled modulate the outline of sRNA-producing loci more than the environment. Notwithstanding the evidence that developmental stage and variety have the strongest effect in terms of distinguishing samples clustering, we were interested to verify the environmental influence on small RNA loci expression in the two cultivars. Thus, for each sRNA-generating cluster we calculated the ratio between cluster abundance in Cabernet Sauvignon and Sangiovese in each environment and developmental stage, thereby revealing the genomic regions with regulated clusters, considering a 2-fold change threshold, a minimum abundance of 5 HNA in each library and a minimum sum of abundance of 30 HNA . Figure 5 shows how different environments affect the production of small RNAs. In Bolgheri, regardless the developmental stage there were many clusters with a very high abundance level in Cabernet Sauvignon . In Montalcino and even more in Riccione we also observed differences between the expressions of clusters in the two cultivars, with ripened and green berries showing an almost opposite profile in terms of number of clusters more expressed in Cabernet Sauvignon or Sangiovese.

When the berries were green, in Montalcino Cabernet Sauvignon shows the highest number of up-regulated clusters, while in Riccione, Sangiovese has the highest number of up-regulated clusters. The opposite behavior was noticed in ripened berries, with Sangiovese having the highest number of up-regulated clusters in Montalcino and Cabernet Sauvignon in Riccione . Notably, we observed a small percentage of regulated clusters exhibiting at least a 10-fold higher abundance of small RNA in Cabernet Sauvignon or Sangiovese when compared to each other . An examination of those clusters showed that a substantial difference could exist between the cultivars, depending on the vineyard and the developmental stage. For example, in Riccione, a cluster matching a locus encoding a BURP domain-containing protein showed a fold change of 390 when comparing green berries of Sangiovese with Cabernet Sauvignon. The small RNAs mapping in this region were mainly 21-nt and produced from both strands . Similarly, the majority of the highly differentially expressed clusters showed a similar profile: strong bias toward 21-nt sRNAs and a low strand bias. These findings suggest that these small RNAs might be the product of RDR polymerase activity rather than degradation products of mRNAs. We applied a pipeline adapted from Jeong et al. and Zhai et al. to identify annotated vvi-miRNAs, their variants, novel species-specific candidates and, when possible, the complementary 3p or 5p sequences. Starting from 25,437,525 distinct sequences from all the 48 libraries, the first filter of the pipeline removed sequences matching t/r/sn/snoRNAs as well as those that did not meet the threshold of 30 TP4M in at least one library or, conversely, that mapped in more than 20 loci of the grapevine genome . Only sequences 18–26-nt in length were retained. Overall, 27,332 sequences, including 56 known vvi-miRNAs, passed through this first filter and were subsequently analyzed by a modified version of miREAP as described by Jeong et al. . miREAP identified 1819 miRNA precursors producing 1108 unique miRNA candidates, including 47 known vvi-miRNA. Next, the sequences were submitted to the third filter to evaluate the single-strand and abundance bias retrieving only one or two most abundant miRNA sequence for each precursor previously identified. A total of 150 unique miRNA corresponding to 209 precursors were identified as candidate miRNAs. Among these 209 candidate precursors, 61 belonged to 31 known vvi-miRNA that passed all the filters and 148 were identified as putatively novel miRNA candidates. To certify that they were novel candidates rather than variants of known vvi-miRNAs we compared their sequences and coordinates with the miRNAs registered in miRBase .

In order to reduce false positives and the selection of siRNA-like miRNAs, we considered only 20, 21, and 22 nt candidates whose stemloop structures were manually evaluated . Eventually, 26 miRNAs homologous to other plant species were identified with high confidence. Twenty-two were new members of nine known V. vinifera families, whereas the other four belong to three families not yet described in grapevine . For 16 homologs we were able to retrieve also the complementary sequence. Finally, excluding these 26 miRNAs and other si-RNA like miRNAs, we identified 7 completely novel bona fide miRNAs. Apart from the 61 known vvi-miRNAs identified by the pipeline, we searched the dataset for others known vvi-miRNAs eliminated throughout the pipeline, looking for isomiRs that were actually more abundant than the annotated sequences. Their complementary 3p or 5p sequence was also retrieved when possible. Hence 89 known vvi-miRNAs were identified in at least one of our libraries . Among the known vvi-miRNAs identified, 24 had an isomiR more abundant than the annotated sequence and 4 have the complementary sequence as the most abundant sequence mapping to their precursor. We found 16 vvi-miRNA isomiRs that were either longer or shorter than the annotated sequence, 7 vvi-miRNAs that mapped in the precursor in a position shifted with respect to the annotated ones and one miRNA that contains a nucleotide gap when compared to the annotated sequence . An extreme case of shifted position was found in vvi-miRNA169c, black plastic planting pots where the annotated sequence had only 5 TP4M when summing its individual abundance in the 48 libraries. Another sequence, shifted 16 bp as compared to its annotated position on the precursor had an abundance sum of 1921 TP4M, and was retained together with the annotated sequence, and named vvi-miRNA169c.1. For 36 of the 48 V. vinifera miRNA families deposited in miRBase we found at least one member. An in silico prediction of miRNA targets was performed for the 191 mature miRNAs here identified. Using the miRferno tool , and considering only targets predicted with high stringency, 1192 targets were predicted for 143 miRNAs, including six completely novel vvi-miRNA candidates . Two novel candidates seem to be involved in the regulation of important secondary metabolites biosynthesis. Among the six targets predicted for grape-m1191, the TT12 gene is known to be involved in the vacuolar accumulation of proanthocyanidins in grapevine . For grape-m1355, 12 targets were predicted and all of them are involved in secondary metabolism pathways. Nine targets code a bifunctional dihydroflavonol 4-reductase that is responsible for the production of anthocyanins , catalyzing the first step in the conversion of dihydroflavonols to anthocyanins . Another targeted gene codes a phenylacetaldehyde reductase which, in tomato, was demonstrated to catalyze the last step in the synthesis of the aroma volatile 2-phenylethanol, important for the aroma and flavor . Still this same miRNA candidate was predicted to target with high confidence a cinnamoyl reductase like protein that is part of polyphenol biosynthetic pathway . The grapem1355 candidate maps on chromosome 3, exactly on the first exon of its target , in a region where another two isoforms of the same gene are located . The last target of this miRNA candidate, codes a cinnamyl alcohol dehydrogenase known to be involved in the lignin biosynthesis . Other novel vvi-miRNA candidates seem to be involved in cell proliferation and in chloroplasts-related functions . Furthermore, for the new vvi-miRC482b candidate, besides the already known involvement of this miRNA family with disease resistance also predicted here, one predicted target encodes an anthocyanin 5-aromatic acyltransferase-like protein known to be involved in the biosynthesis of anthocyanin in different species . As for the conserved known vvi-miRNAs, most of the well-established examples of miR-targets, such as miR156-SPB, miR166-HD-ZIP, miR171-GRAS, miR172-AP2, confirmed in several plant species and already predicted in grapevine, were also predicted here. We studied miRNA profile of accumulation in the different samples. Using their normalized abundance , i.e., their relative cloning frequency, we set an empirical cut off value equal to at least 10 TP4M in both biological replicates to consider a miRNA as expressed in a given library. Also, a miRNA was considered specific when it was expressed in one or more libraries of a unique cultivar, unique environment or unique developmental stage. According to our established cut off, 175 miRNAs were classified as expressed in at least one of our libraries . The libraries constructed from Sangiovese berries at bunch closure collected in Bolgheri showed only 24 expressed miRNAs . For all the other libraries, expressed miRNAs ranged from 76 to 148 . We found very few miRNAs specific to a given condition. The number of specific miRNAs for each cultivar, developmental stage and environment is reported in Figures 8A–C, respectively. Thirty-nine vvi-miRNAs were highly expressed in almost all libraries [21 ubiquitous plus 18 expressed in all libraries except in Bol_SG_bc ], whereas other miRNAs had different accumulation patterns. The normalized expression values of miRNAs were subjected to hierarchical clustering and represented in a heat map . To examine the relatedness among cultivars, environments and developmental stages, we generated a correlation dendrogram . The dendrogram shows, as already suggested by the heatmaps, that a fundamental dichotomy emerges between ripened and green berries. The most evident pattern of expression is observed when comparing different developmental stages, and confirm previous observation of miRNA modulation during fruit ripening . For example, some members of the vvi-miRNA156 family were highly expressed in all ripened berries, but weakly or not expressed in green berries. Differently, vvi-miR396a-3p and vvi-miR396b-3p showed the opposite profile. Similarly, vvi-miR172d, vvi-miR166b-5p, vvi-miR166f-5p, and vvi-miR396d-5p were highly expressed in green berries but weakly expressed in ripened berries and the members of the vvi-miR319 family showed a gradient of decreasing abundance from pea size to harvest.

Avoid placing jars directly on cold surfaces like tile countertops because that may cause jars to crack

Any large covered kettle or pot may be used as a boiling water bath canner, if it is deep enough to allow water to cover the tops of the jars by 1 to 2 inches . Fill the canner half full with water. Preheat the water to 140°F . Place jars on a rack in the canner ; any jars that come in direct contact with the bottom of the canner may break. Add enough boiling water to cover the tops of the jars by at least 1 inch. When water comes to a boil, begin to count the processing time indicated in table 2. At the end of the recommended processing time, remove jars from the canner and cool them, undisturbed, at room temperature. Setting the jars on a towel over the countertop rather than directly on the countertop can help protect jars from breaking. After the jars have cooled, check them for a tight seal. In a tight seal the metal lid will have snapped down and is curved slightly inwards. Press down on the center of the lid. If it springs back, there is no seal—either place this jar in the refrigerator or freezer, or reprocess the contents using a new jar and lid. Remove the rings of the sealed jars and wash the jars gently before storage to remove any stickiness. Washing also removes any excess jam on the jars that may attract insects during storage. Store in a dark, dry, cool place.Presented here is the cumulative research on the response of grapevine within different climate change scenarios, specifically, rising levels of atmospheric carbon dioxide, temperature, and drought. Global warming increases the frequency of extreme high‐temperature events and consequent severe drought scenarios and thus may constitute a threat to modern viticulture . Global warming affects grapevine not only by increasing growing season temperatures, drainage planter pot but also by impacting pest pressure, soil water availability, carbon:nitrogen ratios, and the resulting chemical composition of wine.

Additionally, elevated carbon dioxide causes advances in phenology, which compound significantly over seasons, with the long-term carbon storage increasing after each growing season . Synthesizing the ecological impacts of elevated carbon dioxide on the system of vineyards highlights the profound impact global warming will have on grapevine phenology and subsequent harvest. Grape growers have been keeping detailed records of harvest dates for centuries , and the predicted advances in phenology are creating a challenge for growers. In order for farmers to explore alternative late ripening varieties, we need a quantification of the sensitivity to climate change of international varieties in California. There are between 6,000 – 10,000 genetically different varieties of grapevine . As part of an ongoing research project at the University of California Davis, I recorded the phenological timing of 137 different grapevine varieties and compared this timing across 4 years of varying climate. We modeled the response of the grapevines to temperature and included a variable for days over 40°C to estimate sensitivity to extreme heat. Grouping the varieties by geographic origin and by utility also increased the accuracy of the phenological prediction. This provided perspective on how the vast differences between grapevine varieties contributed to their responses to temperature, and therefore avenues for selective breeding. Many grape growers are legally required to grow specific varieties, which is not the case in California. As a leader in the global market, California could demonstrate the utility of growing alternative varieties as a mitigation to global warming . Furthermore, the commitment to grapevine varieties inspired the research of a targeted genetic transformation to incorporate drought resistance into eminent varieties. One of the mechanisms that plants can activate in response to environmental stresses is the stomatal regulation of transpiration.

The highly conserved hormonal peptides of the epidermal patterning factor family are known in model plants to be responsible for regulating stomatal development during leaf formation . In particular, EPFL9 promotes stomatal development . I studied the role of VvEPFL9 in determining stomatal density in grapevine and determined that stomatal features such as density and distribution are a promising target for designing climate change-resilient crops. Vitis vinifera genotypes with reduced stomatal density and, in turn, greater intrinsic water use efficiency, may in fact be desirable to improve plant water conservation under current and future climate scenarios . The overarching goal of this thesis is to predict the response of grapevines to future climate conditions. The synthesis of current literature on grapevine grown under elevated carbon dioxide levels indicates the major threats include shifts in phenology and drought stress. Modelling the phenological response of 137 varieties over four growing seasons in Northern California created a reference for phenological timing and sensitivity to change in temperature. Furthermore, I transformed grapevine for reduced stomatal density to test the concept of climate change resilient grapevine. I stress that genetic transformation should be used as one tool among many, and this targeted agroecological approach can be used in tandem with exploiting existing grapevine material, which is vast and diverse. Rising atmospheric carbon dioxide levels are well documented by the International Panels on climate change, and carbon dioxide is expected to reach levels between 530 and 720 mg/L by the year 2100 according to intermediate scenarios . The last time Earth experienced levels of carbon dioxide consistently above 400 mg/L was the early Miocene era, approximately 23 million years ago . The earliest agriculture was cultivated between 23,000 and 12,000 years ago , with the earliest grape domestication estimated between 6,000 and 9,000 years ago . Grapevine has historically been sensitive to changes in climate, including the “Little Ice Age” in Europe and the more recent heat waves of the 21st century .

While grapevine is typically cultivated in regions with wet winters and dry summers, increasing events of severe water stress will impede growth and reduce quality and yield in grapevine under climate change . Mean climate projections underestimate the impact of climate change on grapevine, in particular the impact of extreme temperature spikes/drops in areas growing premier winegrapes, currently characterized by few days with extreme heat or cold . While vines in Mediterranean areas will have to adapt to a more variable climate, elevated CO2 will compound the effects of heat and drought stress at a global scale, impacting the quality and quantity of grapevine yield . Carbon dioxide levels present a relatively novel challenge as they have been increasing at an unprecedented rate since the start of the Industrial Revolution . Winegrapes are one of the most culturally and economically important crops worldwide, with an annual production of 60 million tons of fruit annually, the highest monetary value of fruit crops, and wine being part of the UNESCO intangible cultural heritage of humanity . While wild grapevines can be very resilient to abiotic stress, domesticated winegrapes are far more sensitive; a result of the meticulous conservation of berry phenotype with emphasis on flavor over stress tolerance since 400 BC . While this careful preservation of grape berry phenotype benefits the culture and industry of winegrape growing, as an ecological system the vineyard is vulnerable to a changing climate and elevated atmospheric CO2 levels . Heat, elevated carbon dioxide, and limited water availability are necessary for cultivating quality grapes, however, studies on their interactive effects indicate these will have a negative synergistic impact on grapevine . The variety-specific responses to these environmental conditions introduces further variability to any study of grapevine response to future climate , while variability in viticultural production is often viewed as undesirable. The varying physiology of cultivars and the long-term perennial nature of grapevine creates a challenging subject for adaptation studies; we expect that any adaptation will be much slower than that of annual crops . This review synthesizes recent literature published on the direct effects of elevated carbon dioxide on grapevine physiology, as well as the indirect effects on phenology and ecological responses of grapevines, plant pot with drainage including studies of the interactive effects of climate variables. This synthesis focused on literature specific to grapevine, and in addition, included studies on Arabidopsis to explore relevant hypotheses illustrating mechanisms of carbon dynamics in C3 plants. Results were compared from the four predominant experimental approaches; growth chambers, greenhouses, open top chambers, and Free Air CO2 Enrichment , all evaluated for predictive value. Finally, this review concludes by discussing potential research necessary for understanding the future of growing grapevine with elevated CO2 and adaptive viticultural management. The physiological advantage of increased atmospheric carbon available for crops such as grapevine must be weighed against other factors likely to cooccur in the context of climate change, including water scarcity and temperature increases . The literature asserts that the RUBISCO of C3 plants, including grapevines, are currently limited by ambient CO2 substrate and any increases should stimulate carbon assimilation rates and increase vegetative growth , in the absence of other stressors. However, grapevine specific studies provide evidence for down regulation of net photosynthesis as vines acclimate to higher carbon environments .

Salazar-Parra et al. observed a transient increase in maximum photosynthesis in grapevine at elevated CO2, but this effect dissipated over time. A short-term study in a temperature gradient greenhouse at 700 mg/L CO2 showed grapevine photosynthesis increased around the time of veraison , however studies of this duration are more reflective of a high dose of carbon enrichment rather than simulating future climate scenarios. One possible explanation for photosynthetic down regulation, i.e. acclimation, is lowered capacity of the photochemical machinery due to reductions in nitrogen concentrations in the leaf , limiting the activity of the enzyme RUBISCO. Species that are not nitrogen fixing such as grapevine are more likely to experience acclimation in elevated CO2 environments because of limited RUBISCO content . The nitrogen dilution effect is well documented in other crop species, therefore in grapevine, nitrogen use efficiency could increase in elevated CO2 environments because RUBISCO acclimation allows for nitrogen to be redistributed for other growth in the vine, however, FACE experiments documented nitrogen gains lower than predicted . The long-term impact of elevated CO2 on rates of grapevine photosynthesis has been shown to be dependent on other climate factors such as temperature and water availability . Water scarcity, a concomitant climate change variable with elevated CO2, can impact the carbon storage in trunks of vines, as demonstrated in fruit tree orchards, and in turn, drought stress can be partially relieved in elevated CO2 scenarios . Three general physiological responses will benefit grapevine in an elevated CO2 climate with limited water availability; starting with partial stomatal closure limiting water loss, a subsequent increase in soil water content as transpiration decreases, and an increase of starch storage to provide for drought recovery . Acclimation to elevated CO2 will decrease rates of assimilation, while starch reserves increase, as the carbon sink may be driving rates of photosynthesis rather than carbon availability driving metabolism . Therefore, the widespread observed reduction in stomatal conductance and density may have a greater impact on grapevine water use efficiency from decreasing transpiration rather than increasing carbon assimilation. In the past ten years, grapevine physiology research under elevated CO2 has focused on the impacts on WUE defined as carbon assimilated per unit of water transpired. Grapevine relies on stomatal aperture to facilitate cooling and CO2 uptake, releasing latent heat as the plant reaches physiological temperature thresholds; however, closure is essential to avoid detrimental water loss, heat damage, and reduced photosynthate production . With higher levels of carbon dioxide in the atmosphere, stomata can facilitate a lower water per CO2 molecular exchange, increasing the leaf level WUE . An early study of grapevine under elevated CO2 treatment for one season found no significant effect on stomatal conductance and transpiration . Subsequently, a study using 650 mg/L in a similar open top chamber treatment found gs and transpiration decreased at elevated CO2 . In contrast, at only at 500 mg/L, higher gs and transpiration rates were observed in grapevines in a consistently elevated CO2 environment for three consecutive seasons . On a morphological level, multiple studies have documented the reduction in stomatal density in several varieties of grapevine . Scaling intrinsic water use efficiency to the whole plant level will require documenting changes in microclimate as well as morphology, such as stomatal density and leaf area .

The result is a skillful revision of American history as a plainly imperialist enterprise

The last decade alone has witnessed the emergence of literary, postcolonial, and other theoretical perspectives in studies of space and place. Other geographers now study maps and contemporary geographic information systems and how both have been used by Natives and non-Natives to represent Indian and Inuit places. Still others engage directly in advocacy on behalf of Indian economic development and land claims. In what follows, however, readers also will find direct links to Sauerian-Boasian geographies of American Indians via cultural ecology, diffusion studies, and regional-historical geography. Recently geographers have contributed significantly to revisionist movements within North American cultural ecology, illuminating the sophistication of American Indian environmental management and the degree to which people intentionally modified environments for their own purposes prior to European contact. For example, two geographers identified pre-contact plant cultivation practices and agricultural land forms that had been overlooked by many archaeologists and anthropologists, namely the identification of ridged agricultural fields and other evidence of intensive environmental management in the upper Midwest. Others have expanded our understanding of human modification of environments in the American southwestern deserts, Alaska, Wisconsin, and central California. Also, Douglas Deur has demonstrated the presence of low-intensity plant cultivation in garden plots on the west coast of Canada, a region usually depicted as non-agricultural. Janet Gritzner and Sandra Peacock, working just east of Deur in the semi-arid interior plateaus of both the United States and Canada, have shown that the peoples of this region managed plants intensively through prescribed burning, selective harvesting, and the transporting and transplanting of plant materials. Karl Butzer and William Denevan have produced syntheses summarizing Indian impacts on the land throughout North America, drainage pot and have demonstrated the value of this environmental modification to later European occupation of the continent.

These studies often integrate bio-geography, or the study of plant and animal distributions and movement, analysis of settlement patterns and archaeological features, and conventional ethnographic methods, thereby shedding light on related topics such as pre-contact Native settlement distribution, population size, and cultural complexity. For the post-contact period, interpretations of the fur trade and other similar networks have been overhauled by a new focus on Indians as active trade agents, modifiers of landscapes, and manipulators of markets and geopolitics. Returning to the continental scale once again, historical geographer Donald Meinig has reinterpreted the shaping of the United States by synthesizing trade research and other information on Indian networks with research on US religious, corporate, and military interests aiming to secure a continent-wide economic and cultural empire. Another persisting theme associated with Berkeley geography entails explaining the origins and diffusion pattern of specific elements of the material cultural landscape. For example, the following have appeared in recent years: a regional analysis of material landscapes in the Four Corners area; a typology of Canadian Metis houses and farmsteads; a comparative study of gravesites and outdoor funerary practices among the Navajo, Mormons, and Zuni; examinations of Mescalero Apache settlement and housing; analyses of architecture and its geographical context; a study of conflicting cultural landscapes at Santa Clara Pueblo by a member of that pueblo; and a diffusion study supporting the thesis that there was early Asian contact with Indians in the pre-Columbian Americas, an idea still not widely accepted in geography. In another materialist study, Terry Jordan and Matti Kaups argued that early New Sweden produced a successful mix of Finnish and Delaware Indian forest cultures in the Delaware Bay area that then expanded into the Upland South, becoming the most successful frontier society in early America. However, one critic was chagrined that the authors had handled questions of backwoods relationships, intermarriage, and other social customs only from the European perspective.

The material elements of the biosphere normally termed resources might also be worth considering simply as sources. So many issues associated with the biosphere, like water quality and quantity, are deeply intertwined with sovereignty, land dispossession and restoration, planning and development, sacredness, and even gambling and tourism. All these topics are sources of physical, economic, political, and spiritual livelihoods. While this manipulation of language may seem thoroughly postmodern, it is not our intention to be playful. We employ ambiguity as a linguistic element expressing the intellectual richness of this subject by acknowledging the inter-penetration of its subtopics. In this section then, we seek to dissolve the traditional human-nature dichotomy by grouping these ideas together and employing resource and source as one word. Since 1980 the capacity of tribal governments as sovereign managers of sources and environmental quality has increased modestly in some cases, dramatically in others. Individual tribal members working as attorneys for or leaders of tribal governments or pan-Indian organizations such as the Native American Rights Fund and the Council of Energy Resource Tribes frequently raise their voices to characterize human-environment relations from an Indian perspective. Geographers studying tribal source management are responding to the same contemporary Native issues raised in these settings. Water has been of special interest to those working in the and and semiarid western United States. In this area, water not only is physically and economically crucial, but also is an agent of social cohesion and cultural reaffirmation. Kate Berry demonstrated this in two recent publications comparing the role of water for two Paiute communities and one non-Indian community in northwestern Nevada, and the role of water in the historical geography of California. Berry and Joanne Endter also examined the significance of different cultural values in the actions, policies, and court decisions affecting water allocation for tribes.

Judith Jacobsen and Mary McNally considered how water project development and marketing of water rights on tribal land has been influenced by congressional actions and negotiated water rights settlements, and John Newton looked at local responses to flood hazard in northern Canada.“ Noteworthy contributions also have been made in the analysis of tribal control over plant and wildlife sources. The Clayoquot Sound Scientific Panel in British Columbia, a group including both Indian and non-Indian researchers, articulated how traditional understandings and uses of forests worked in the pre-European occupation period and demonstrated how such perception and knowledge might be accommodated in current land management practices.“ Steven Silvern studied tribal control of off-reservation fish and wildlife sources guaranteed by treaty by examining the structure of conflict between hunters and fishers and six Chippewa tribes in Wisconsin. The infamous government-ordered Navajo sheep-reduction program of the 1930s may stand alone as the worst example of the government’s willingness to pauperize a population by eliminating its animals. Will Graf and others confirmed that where Indian-white relations are concerned, “a little scientific knowledge [is] a dangerous commodity.” Graf, a physical geographer, states that the Navajo were right in asserting that climate variation was the cause of Colorado River sedimentation, not the number of sheep they grazed. The Bureau of Reclamation and Soil Conservation Service were comfortable nonetheless in recommending the reduction program when they had only incomplete data. In jarring contrast, large pot with drainage co-management agreements for wildlife and other biota are so common across Canada now that an argument has been made for including them as part of the constitutional rights of First Nations. A key element in sovereignty disputes is the relationship of land claims and land rights to contemporary tribal powers and land use decision-making. Sovereignty is also central to the expansion of tribal powers and autonomy within states, provinces, and federal governments. In particular, the recognition of tribal governments as legitimate controlling agents of tribal sources has put the sovereignty issue on center stage among geographers and planners. For example, Dick Winchell described the concept of inherent sovereignty as the basis of tribal powers while also offering ways of incorporating inherent sovereignty into contemporary comprehensive planning models. In addition, David Wishart recognized that sometimes contemporary reservations are good bulwarks against further attacks on sovereignty. Sovereignty issues are implicated in “dispossession research,” which is conducted at all geographic scales. Perhaps no one has summarized in a single map the continent-wide geography of Indian removals during the colonial period better than Elaine Bjorklund. For specific regions, there are Kenneth Brealey’s study of the genesis of British Columbia’s reserve system, Malcolm Comeaux’s description of how water and development economics were used to dispossess the Pima and Maricopa, Florence Shipek’s volume on southern California, where Indians were literally pushed into the rocks, and Wishart’s book detailing the elimination of people from nineteenth-century Nebraska.

The latter two are the culmination of decades of archival research and, in Shipek’s case, ethnographic fieldwork. Dispossession also continues in its classic form today, as Ward Churchill summarized for a number of places, and as Holly Young bear-Tibbetts described in detail for the White Earth Anishinaabeg. Dispossession is more than a physical act, for it occurs in rhetorical strategies that anticipate the action. Randy Bertolas examined such a strategy in the redefinition of Cree places as “wilderness.” He argued that imagining a place as empty of humans, although only a dream, allows the colonizer-dreamer to then separate people from their own socially constructed landscapes, causing seemingly less psychic pain for the colonizer. Robert Bone described essentially the same process for the Canadian North.30 There are newer or less commonly used methods too, including gerrymandering a reservation to reduce electoral rep resentation, and contesting tribal rights to select an electricity provider by going to court to call into question the “Indianness” of a reservation. Imre Sutton, a political geographer, summarized the historical evolution of Indian Country as a site of legal jurisdiction in substantial detail. Dorothy Hallock gave a more abbreviated summary, but recast contemporary Indian Country as the location of numerous aspects of colonizer-colonized relations, not just as the seat of Indian legal claims. She also suggested that Indian Country was a general geographical model for all majority-minority relations. Finally she reported a case study out of her own experience as a planner on the Fort Mojave Reservation, part of which was redesigned as an internment camp for Japanese-Americans during World War Ⅱ. Canadian geographers seem to have given more thought to the basis for land claims and their potential resolution, a matter probably arising out of the contemporary political circumstances in which Canadians find themselves. Taking an economic approach, Frank Duerden looked at land claims in the Yukon as a land location-allocation problem, Evelyn Peters drew lessons from the Cree situation in Quebec, and Peter Usher et al. provided a valuable and detailed diachronic summary of tenure systems, Indian and Inuit concepts of rights, and ongoing claims in British Columbia and the Arctic. Geographers are by no means uniformly aligned in their political sentiments regarding Indian land claims. Political agendas and associated ideologies vary widely and are often evident in what they write and whom they consult. Some in the United States still willfully disregard Indian land rights when staking out their own positions in support of a competing land claim, despite extensive writing on the subject by Linda Parker, Sutton, and others cited above. For example, the January 1999 issue of Political Geography included a debate over the future of the Hanford Plant just across the Rattlesnake Hills from the Yakima Reservation in south central Washington. A respected political geographer wrote that Yakima rights were only those of a “local interest group” that had not exercised these “local customs” in the more than fifty years since Hanford was built. Several critics attempted to set the writer straight, but no acknowledgement of their criticisms was registered in his rejoinder. Out of the claims process can come land restorations, although it is highly uncommon in the United States. Barbara More house recounts the success of the Havasupai in regaining trust over 185,000 acres of Grand Canyon National Park. Five other tribes gained very little, but More house’s analysis suggests that the Havasupai were more successful not because of longevity, occupancy, or other similar evidence, but because they broadened their rhetorical arguments beyond the narrow concerns of the moment to include most of the other Grand Canyon interests, both Indian and non-Indian, involved in that negotiation. More house leaves unanswered the question of whether such rhetoric has an historical basis in Havasupai culture or if it represents a form of “talking back by using one of the colonizer’s own argument strategies. Another aspect of the colonial legacy is the need to document sacred land.

Simple theoretical models that produce quantized anomalous Hall effects have been known for decades

Similarly, attaching a Chern magnet to a reservoir of electrons and using an electrostatic gate to draw electrons into the magnet will populate additional chiral edge states. Properties that depend on the number of electrons occupying these special quantum states will change accordingly. In all of these systems, conductivity strongly depends on the number of quantum states available at the Fermi level. For metallic systems, the number of Bloch states available at any particular energy depends on details of the band structure. The total conductance between any two points within the crystal depends on the relative positions of the two points and the geometry of the crystal. Thus conductivity is an intrinsic property of a metal, but conductance is an extrinsic property of a metal, and both are challenging to compute precisely from first principles. When the Fermi level is in the gap of a Chern magnet, there exists a number of quantum states at the Fermi level exactly equal to the Chern number. The conductance through a small number of delocalized quantum states is quite generally equal to e 2 h per quantum channel, and so the conductance between two electronic reservoirs in contact with the same edge of a Chern magnet is equal to C e 2 h . These facts together make the conductance and not just the conductivity an intrinsic property of a Chern magnet. This is a remarkable fact; indeed, as long as a researcher has access to a tool for measuring electrical resistance and a Chern magnet, they can directly measure a combination of the fundamental constants e and h through a simple electronic transport experiment- they don’t need to know anything about the geometry or band structure of the Chern magnet. This phenomenon is known as the quantized anomalous Hall effect. Chern magnets often support magnetic hysteresis, just like trivial magnetic insulators. Because the chirality of the edge state is determined by the sign of the Chern number, greenhouse vertical farming and the sign of the Chern number is determined by the bulk magnetization, quantized anomalous Hall effects usually exhibit magnetic hysteresis.

The quantized anomalous Hall effect is so unique to Chern mangets that that it is sometimes used as to define the entire class of systems; i.e., researchers historically have described these magnets as ‘QAH materials,’ or ‘QAH magnets.’ At finite temperature, electrons occupying Bloch states in metals can dissipate energy by scattering off of phonons, other electrons, or defects into different nearby Bloch states. This is possible because at every position in real space and momentum space there is a near-continuum of available quantum states available for an electron to scatter into with arbitrarily similar momentum and en- ergy. This is not the case for electrons in chiral edge states of Chern magnets, which do not have available quantum states in the bulk. As a result, electrons that enter chiral edge state wave functions do not dissipate energy. There is a dissipative cost for getting electrons into these wave functionsthis was discussed in the previous paragraph- but this energetic cost is independent of all details of the shape and environment of the chiral edge state, even at finite temperature. This is why the Hall resistance Rxy in a Chern magnet is so precisely quantized; it must take on a value of 1 C e h 2 , and processes that would modify the resistance in other materials are strictly forbidden in Chern magnets. All bands have finite degeneracy- that is, they can only accommodate a certain number of electrons per unit area or volume of crystal. If electrons are forced into a crystal after a particular band is full, they will end up in a different band, generally the band that is next lowest in energy. This degeneracy depends only on the properties of the crystal. Chern bands have electronic degeneracies that change in response to an applied magnetic field; that is to say, when Chern magnets are exposed to an external magnetic field, their electronic bands will change to accommodate more electrons. The challenge, then, lay in realizing real materials with all of the ingredients necessary to produce a Chern magnet. These are, in short: high Berry curvature, a two-dimensional or nearly two-dimensional crystal, and an interaction-driven gap coupled to magnetic order. It turns out that a variety of material systems with high Berry curvature are known in three dimensions; three dimensional topological insulators satisfy the first criterion, and are relatively straightforward to produce and deposit in thin film form using molecular beam epitaxy, satisfying the second.

These systems do not, however, have magnetic order. Researchers attempted to induce magnetic order in these materials with the addition of magnetic dopants. It was hoped that by peppering the lattice with ions with large magnetic moments and strong exchange interactions that magnetic order could be induced in the band structure of the material, as illustrated in Fig. 3.11. This approach ultimately succeeded in producing the first material ever shown to support a quantized anomalous Hall effect. An image of a film of this material and associated electronic transport data are shown in Fig. 3.12.There are many a priori reasons to suspect that magnetically doped topological insulators might have strong charge disorder. The strongest is the presence of the magnetic dopants- dopants always generate significant charge disorder; in a sense they are by definition a source of disorder. Because their distribution throughout the host crystal is not ordered, dopants can reduce the effective band gap through the mechanism illustrated in Fig. 3.14. It turns out this concern about magnetically doped topological insulators has been born our in practice; the systems have been improved since their original discovery, but in all known samples the Curie temperatures dramatically exceed the charge gaps . This puts these systems deep in the kBTC > EGap limit. The resolution to this issue has always been clear, if not exactly easy. If a crystal could be realized that had bands with both finite Chern numbers and magnetic interactions strong enough to produce a magnetic insulator, then we could expect such a system to be a clean Chern magnet . Such a system would likely support a QAH effect at much higher temperature then the status quo, since it would not be limited by charge disorder. We now have all of the tools we need to begin discussing real examples of Chern magnets on moir´e superlattices. Our discussion will begin with twisted bilayer graphene. We have already discussed the notion that moir´e superlattices can support electronic bands, and that we can expect these bands to accommodate far fewer electrons per unit area than bands in atomic lattices . This was pointed out in 2011 by Rafi Bistritzer and Allan MacDonald, but they also made another interesting observation: the band structure of the moir´e superlattice is highly sensitive to the relative twist angle of the two lattices, and the bandwidth of the resulting moir´e bands can be finely tuned using the relative twist angle as a variational parameter. It turns out twisted bilayer graphene moir´e superlattice bands can be made to have vanishingly small bandwidth by tuning the twist angle to the so-called ‘magic angle.’

The magic angle is around 1.10- 1.15◦ and a schematic of magic angle twisted bilayer graphene is shown in Fig. 4.2A. The computed band structure of twisted bilayer graphene is illustrated in Fig. 4.2B for a few different twist angles, including the magic angle. The other bands are grayed out at the magic angle to illustrate the low bandwidth of the moir´e superlattice bands. This is an interesting situation for a variety of reasons we have already covered. The low bandwidth of the moir´e superlattice bands combined with the low electronic density required to fill them makes them especially appealing targets for electrostatic gating experiments. The system is relatively easy to prepare; twisted bilayer graphene devices are produced by ripping a monolayer of graphene in half, rotating one crystal relative to the other using a mechanical goniometer, and then overlaying it on the other. The ‘flatness’ of the band also makes this system especially likely to support interaction-driven electronic phases like magnetism or superconductivity . It is also worth mentioning that it is extremely easy to identify situations in which interactions produce gaps in these systems. Because gaps appear when the moir´e superlattice bands are completely filled with electrons or with holes, nft vertical farming and we already know that the moir´e superlattice bands are fourfold degenerate, we can expect any interaction-driven insulating phase to appear as an insulating phase at precisely 1/4, 1/2, or 3/4 of the electron density required to reach full filling of the moir´e superlattice band. These are sometimes called ‘filling factors’ of 1, 2, and 3, respectively, referencing the number of electrons per moir´e unit cell. This argument is presented in schematic form in Fig. 4.2 in the context of experimental data. Interaction-driven gaps were first discovered in 2018, and this discovery was quickly followed by the dramatic discovery of superconductivity in twisted bilayer graphene. Other researchers predicted that breaking inversion symmetry in graphene would open a gap nearcharge neutrality with strong Berry curvature at the band edges. The graphene heterostructures we make in this field are almost always encapsulated in the two dimensional crystal hBN, which has a lattice constant quite close to that of graphene. The presence of this two dimensional crystal technically always does break inversion symmetry for graphene crystals, but this effect is averaged out over many graphene unit cells whenever the lattices of hBN and graphene are not aligned with each other. Therefore the simplest way to break inversion symmetry in graphene systems is to align the graphene lattice with the lattice of one of its encapsulating hBN crystals. Experiments on such a device indeed realized a large valley hall effect, an analogue for the valley degree of freedom of the spin Hall effect discussed in the previous chapter, a tantalizing clue that the researchers had indeed produced high Berry curvature bands in graphene. Twisted bilayer graphene aligned to hBN thus has all of the ingredients necessary for realizing an intrinsic Chern magnet: it has flat bands for realizing a magnetic insulator, it has strong Berry curvature, and it is highly gate tunable so that we can easily reach the Fermi level at which an interaction-driven gap is realized. Magnetism with a strong anomalous Hall effect was first realized in hBN-aligned twisted bilayer graphene in 2019. Some basic properties of this phase are illustrated in Fig. 4.3. This system was clearly a magnet with strong Berry curvature; it was not gapped and thus did not realize a quantized anomalous Hall effect, but it was unknown whether this was because of disorder or because the system did not have strong enough interactions or small enough bandwidth to realize a gap. The stage was set for the discovery of a quantized anomalous Hall effect in an intrinsic Chern magnet in hBN-aligned twisted bilayer graphene. An optical microscope image of the tBLG device discussed here is shown in Fig. 4.4A. The device is made using the “tear-and-stack” technique, in which one half of a graphene monolayer is torn off, rotated by a precise relative alignment angle , and then placed on top of the other half of the monolayer. The tBLG layer is sandwiched between two hBN flakes with thickness 40 and 70 nm, as shown in Fig. 4.4B. A few-layer-thick graphite flake is used as the bottom gate of the device, which has been shown to produce devices with low charge disorder . The stack rests on a Si/SiO2 wafer, which is also used to gate the contact regions of the device. The stack was assembled at 60C using a dry-transfer technique with a poly film on top of a polydimethylsiloxane stamp. In an exfoliated heterostructure, the orientation of the crystal lattice relative to the edges of the flake can often be determined by investigating the natural cleavage planes of the flake. Graphene and hBN, being hexagonal lattices, have two easy cleavage planes – zigzag and armchair, each with six-fold symmetry, that together produce cleavage planes for every 30relative rotation of the lattice. We tentatively identify crystallographic directions by finding edges of the flakes with relative angles of 30. From the optical image we find that the cleavage planes of the tBLG layer and the top hBN are aligned.

The second effect cannot be replicated in three dimensional systems with any known technique

We suggest research and development efforts in the exploration of a combined certification approach , which could balance the costs and benefits of different certification systems . Because certification can be expensive, multiple certifications may be cost prohibitive, especially for smallholder farmers , but discounts or incentives could be put in place in order to minimize the costs of multiple certifications. Alternatively, government agencies could subsidize or provide loans for the initial costs of certification and transition, or these expenses could be paid after the first years’ profit are earned. In this way, the certification system could be revised to be more inclusive of small landholders. It is also essential that certification studies incorporate an analysis of the time, labor, and economic costs involved. In future work, the support needed from financial, institutional, and community agencies in order to successfully transition non-certified farmers to organic, fair trade, or biodiversity- or livelihood-friendly coffees should be explicitly investigated.Since 1989, the role of national governments directly influencing global coffee markets and prices paid to producers has decreased , and, in these years, in many regions, rural poverty rates have increased together with accelerating rates of environmental destruction . We suggest that national governments of coffee-producing regions need to play a more active role in providing basic services to their populaces and in protecting ecosystem services. Payments for ecosystem services provide one avenue for compensation from the beneficiaries directly to the landholders and have been implemented in a number of nations, including Costa Rica, Mexico, and China . Rewards for ecosystem services should not be used to directly regulate land management, raspberry cultivation pot but they could provide valuable incentives, especially with the incorporation of management extension services .

The difficulties of quantifying payments for ecosystem services or integrating them with the practices of potential stakeholders or government agencies create real challenges . Therefore, successful programs require stakeholder involvement and development of sustainable farmer livelihoods . Local, regional, and even national cooperatives with administrative capacity and accountability to their membership can leverage international development funding to improve coffee yields and quality, increase production from the diversified shade canopy, and support a wide array of social development projects . Incentives and infrastructure directed toward farmers who use sustainable practices and preserve biodiversity could encourage producers to be good stewards of the land while making a living.Our findings show that, although global coffee acreage has decreased since 1990, cultivation has grown dramatically in Asia and has been accompanied by declining levels of diverse-shade coffee, which threatens the availability and flow of ecosystem services across the globe. Although there have been several gains in the growth of sustainable certifications, research also suggests that livelihoods remain vulnerable and that poverty and hunger are persistent in many farming communities. Research in coffee systems has allowed for an improved understanding of habitat management and biodiversity, a closer examination of the relationships between biodiversity and ecosystem services, and a greater understanding of tropical spatial ecology and connectivity. Coffee has also emerged as an important test case for assessing the effects of different certification programs, evaluating the links between local and global economies, and examining the arena for participatory and interdisciplinary research. However, diversified efforts are needed to develop effective solutions for sustainable livelihoods, and it is essential that all members in the coffee value chain become active stakeholders in these efforts.

From local to global scales, it is clear that farmers, cooperatives, government agencies, and consumers all influence coffee land management and rural livelihoods. We have documented that, in many of the landscapes that generate important ecosystem services, the benefits are not necessarily harvested in terms of income, incentives, and opportunities. In order for coffee landscapes to be sustainable for humans and their ecosystems, we need to better incorporate human well-being and livelihoods into global concepts of sustainability,  encourage the diversification of coffee farms to promote greater resilience to changes in global markets and climates, and improve the valuation and reward for ecosystem services through certification and other systems in order to compensate farmers for the innumerable benefits that shaded landscapes provide. Building synergistic and cooperative relationships among farmers, certifiers, global agencies, researchers, and consumers can provide greater transparency and creative solutions for promoting ecological processes and well-being across global coffee systems.One can achieve dramatic changes in charge density using this technique, but that comes with a heavy cost- the crystal is no longer uniform, as every dopant contributes to disorder, and at high doping levels the band structure itself can be modified by the dopant atoms. More important than all of this for the purposes of experimental physics, however, is that under most circumstances the dopant concentration within a crystal can only be modified through laborious chemical treatments of a particular sample. Materials scientists working under these constraints who wish to explore electron density as an independent variable must either find ingenious material-specific techniques for modifying the dopant concentration in situ , or else they must make a separate sample for each data point they would like to present in their experiment. This is an incredibly labor-intensive process, and it also comes with another significant downside: comparing the properties of two different samples with different doping densities exposes results to systematic differences in sample geometry and imperfections in protocol repeatability, and it is difficult to deconvolute these from the effects of changes in electron density.

For these reasons electron density has generally been an awkward and labor intensive independent variable to manipulate. I have found that there are a few ideas that occur naturally to newcomers and outsiders to the field that insiders know enough to immediately discount, and I’d like to discuss one of those ideas here. Chemical doping to manipulate electron density is an ingenious and important technique, but suppose we tried something much sillier- suppose we simply forcefully deposit electrons onto a crystal using some mechanical or electrical process. Would this not achieve our goal? In fact this does indeed work, we have machines that can do this- van de Graff generators can deposit charge onto a piece of metal mechanically, and a variety of other machines can mimic this behavior electronically. So why aren’t condensed matter physicists going around gluing interesting crystals to van de Graff generators so that we can controllably charge them up and measure their responses to changes in charge density? There are a few reasons, but the most important one is that there is a fundamental issue with manipulating charge density this way in three dimensions: this process does not produce a uniform distribution of electron density within the crystal we’d like to study. In three dimensional systems subjected to this treatment, as illustrated in Fig. 1.1B, excess charge accumulates on the surfaces of the crystal, and although we can force additional electrons into acrystal this way we do not ultimately get a system with a modified but still uniform electron density for us to study. This is not the case for two dimensional systems. Those readers with any exposure to introductory physics have likely encountered parallel plate capacitors; these are highly idealized systems composed of a pair of infinitely thin conducting sheets separated by a small insulating space of consistent thickness. When a voltage is applied to one of these sheets with the other connected to a reservoir of mobile electrons, a uniform charge density per unit area appears on both sheets . Of course, low round pots in real metallic capacitors the charge density per unit volume is often still not microscopically uniform because the sheets are not actually infinitely thin, so electrons can redistribute themselves in the out-of-plane direction. To achieve true uniformity one of the plates of the capacitor must be atomically thin, so that electrons simply cannot redistribute themselves in the out-of-plane direction in response to the local electric field. An efficient technique for preparing atomically thin pieces of crystalline graphite was discovered in 2004 by Dr. Andre Geim and Dr. Konstantin Novoselov, an achievement for which they shared the Nobel prize in physics in 2010. The technique involves encapsulating a crystal within a piece of scotch tape and repeatedly ripping the tape apart; it works because the out-of-plane bonds in graphite are much weaker than the in-plane bonds. Graphite represents something of an extreme example of this condition, but it is satisfied to varying extents by a large class of other materials, and as a result the technique was rapidly generalized to produce a variety of other two-dimensional crystals. By constructing a capacitor with one gate replaced with one of these two dimensional crystals, as shown in Fig. 1.1D, researchers can easily access electron density as an independent variable in a condensed matter system. These systems also facilitate an additional degree of control, with no real analogue in three dimensional systems. By placing capacitor plates on both sides of the two dimensional crystal and applying opposite voltages to the opposing gates, researchers can apply out-of-plane electric fields to these systems . A semiclassical model- in which electrons within the system redistribute themselves in the out-of-plane direction to screen this electric field- does not apply; instead, the wave functions hosted by the two dimensional crystal are themselves deformed in response to the applied electric field .

This changes the electronic band structure of the crystal directly, without affecting the electron density. So to summarize, when a two dimensional crystal is encapsulated with gates to produce a three-layer capacitor, researchers can tune both the electron density and the band structure of the crystal at their pleasure. In the first case, this represents a degree of control that would require the creation of many separate samples to replicate in a three dimensional system. There is a temptation to focus on the exotic phenomena that these techniques for manipulating the electronic structure of two dimensional crystals have allowed us to discover, and there will be plenty of time for that. I’d first like to take a moment to impress upon the reader the remarkable degree of control and extent of theoretical understanding these technologies have allowed us to achieve over those condensed matter systems that are known not to host any new physics. I’ve included several figures from a publication produced by Andrea’s lab with which I was completely uninvolved. It contains precise calculations of the compressibility of a particular allotrope of trilayer graphene as a function of electron density and out-of-plane electric field based on the band structure of the system . It also contains a measurement of compressibility as a function of electron density and out-of-plane electric field, performed using the techniques discussed above . The details of the physics discussed in that publication aren’t important for my point here; the observation I’d like to focus on is the fact that, for this particular condensed matter system, quantitatively accurate agreement between the predictions of our models and the real behavior of the system has been achieved. I remember sitting in a group meeting early in my time working with Andrea’s lab, long before I understood much about Chern magnets or any of the other ideas that would come to define my graduate research work, and marvelling at that fact. Experimental condensed matter physics necessarily involves the study of systems with an enormous number of degrees of freedom and countless opportunities for disorder and complexity to contaminate results. Too often work in this field feels uncomfortably close to gluing wires to rocks and then arguing about how to interpret the results, with no real hope of achieving full understanding, or closure, or even agreement about the conclusions we can extract from our experiments. Within the field of exfoliated heterostructures, it is now clear that we really can hope to pursue true quantitative accuracy in calculations of the properties of condensed matter systems. Rich datasets like these, with a variety of impactful independent variables, produce extremely strong limits on theories. They allow us to be precise in our comparisons of theory to experiment, and as a result they have allowed us to bring models based on band structure theory to new heights of predictive power. But most importantly, under these conditions we can easily identify deviations from our expectations with interesting new phenomena- in particular, situations in which electronic interactions produce even subtle deviations from the predictions of single particle band.

Its population dynamics over time are therefore expected to be both oscillatory and unpredictable

As is frequently noted, the green coffee scale insect is a persistent but relatively benign coffee herbivore, only rarely reaching important pest status, although with clear potential to do so . A casual walk on a coffee farm reveals what appears to be a regulating factor. Searching coffee bushes, one finds scale insects here and there and, importantly, a small species of beetle that is evidently feasting on them . The predatory beetle is Azya orbigera, in the family Coccinelidae. Without a doubt, this observation can easily lead to the conclusion that the relatively rare scale insect is kept under control by the relatively common coccinellid beetle. But a closer look reveals a dramatic variability: Some bushes are very heavily laden with the scale insects, and some have none at all. There is another classical ecological notion that emerges in this system. Surrounding the tree in which an Azteca nest is located is a region containing coffee plants that are routinely patrolled by the Azteca ants that were described above. The ants harvest the sweet secretions the scale insects produce and, in turn, scare away or kill the natural enemies seeking to attack the scales , a well-known mutualism . Because the coffee bushes located near the shade trees that contain Azteca nests are where the scale insect is at least partially protected from the predatory beetle and various parasitoids, this area represents a refuge for the scale insect. It is therefore tempting to conclude that the ant itself is an indirect herbivore on the coffee . Although such is the case at a very local level , because of the complexities induced by the beetle predator, such is not the case at a larger scale. The ants effectively provide an area of high food availability for the beetle. Furthermore, the ants protecting the scale insects also, inadvertently, dutch bucket for tomatoes protect the beetle larvae from its own parasitoids, providing an effective refuge for the beetle as well .

Predator–prey systems that contain a refuge are well studied in theoretical ecology , usually with an emphasis on their stabilizing properties. Expanding our view to a larger spatial scale, we deduce an evident contradiction from easily observable patterns. The scale insects are inevitably eaten by the predatory beetle unless they are protected by the ants. However, the ants cannot provide protection if they have not yet created a foraging pattern at the site where the scales are located. Therefore, the scale insect is unable to form a successful population unless under protection from the ants but is unable to attract the ant protection unless it builds up at least a small population. This pattern is well known in ecology as an Allee effect: An organism cannot form a successful population unless a critical number of individuals first become established, a mechanism generally understood to frequently be involved with the idea of critical transitions. In figure 4, we illustrate the system with a cartoon diagram approximately summarizing a simple population model . On one hand, as the dispersion of scales moves from a position far removed from the refuge toward it, the adult beetle predators that have already located the scales will tend to move with it, until they encounter the protective ants , as is presented in figure 4a. A snapshot at some particular time therefore might look like the pattern in figure 4b. On the other hand, as the dispersion of scales moves from a position within the refuge away from it, the encounter with the beetle predators will not occur until the scales are far removed from the refuge, as is presented in figure 4c. A snapshot at some particular time therefore might look like the pattern in figure 4d. Finally, combining the pattern of figure 4b with that of figure 4d, we obtain the combined graph presented in figure 4e. Note that there is a broad region in which the scales could be very high while at the same time could be very low, effectively depending on where the scales are dispersing from, a structure typically referred to as hysteresis. Selecting 20 different shade trees containing Azteca nests, we examined all coffee bushes within 2 meters of the nest and a number of bushes further removed .

We estimated the activity of Azteca ants on each of the bushes before counting the scale insects, to get an estimate of where the actual refuge was located . Note that the ant activity within 1 meter of the nest was high for almost all bushes surveyed , although positions greater than 1 meter awaty were highly variable, with some bushes having high activity levels and others having none. Further than 4 meters from the nest, ant activity was effectively nonexistant, and bushes further than about 4 meters from the nest were completely out of the refuge. Plotting the number of bushes with a saturated density of scale insects and those with less than 10 scales, we obtain a pattern corresponding quite closely to what is expected from the hysteretic pattern predicted by the theoretical considerations . A further complication enters with a more complete natural history understanding of the beetles and their larvae. Although the adult beetle can fly and therefore forage over long distances for its food source, the larvae are largely restricted to terrestrial movement; that is, they are restricted in space . Female beetles therefore must choose their oviposition sites in such a way that the larvae will mature in an environment that contains a locally abundant food source. One major food source for predatory beetles is the general kinds of insects that are relatively sessile and suck the juices from plants, precisely the characteristics of the green coffee scale. They are easy targets for predators because they are normally slow moving and have few defenses. The problem for a potential predator is that they are very frequently defended by ants, precisely in areas where they are good sources of food for a beetle larva. Consequently, a whole group of beetles has evolved the habit of seeking out ants and ovipositing in areas where ants are abundant and defending the hemipterans. These myrmecophilous beetles must obviously have a strategy of protecting their larvae from the aggressive action of the ants and of enabling oviposition in sites of high ant activity . In the case of the beetle A. orbigera, the larva is covered with waxy filaments that tend to stick in the ants’ mandibles whenever they try to attack it .

But more importantly, female beetles take advantage of an unusual behavioral pattern of the ants in order to oviposit where the scales are abundant . When a phorid fly attacks an ant, that ant exudes a pheromone that effectively says to the other ants in the general vicinity “Look out! Phorids attacking,” and the surrounding sisters all adopt a sort of catatonic posture, heads up, mandibles open, and stationary . Although the phorid is able to detect the alarm pheromones of the ant and is therefore attracted to it, it is unable to actually oviposit on the ant unless it sees some movement . Therefore, not only the ant under potential phorid attack, but also the sisters surrounding her assume this semistationary posture, a result of the very specific pheromone that alerts all ants in the vicinity that a phorid is lurking about. Remarkably, the adult female beetle is able to detect and react to this specific chemical, apparently using it as a cue that the time is propitious to enter into the ant-protected zone to sneak in some ovipositions . Therefore the phorid, in addition to being an important player in the Turing process that forms the basic spatial structure of the system, imposes a trait-mediated indirect interaction , in which the effect of the ant on the beetle is reduced. There is more to this story: first, from simple theoretical considerations and, second, from some evident natural history observations of the system. The theoretical considerations emerge from the knowledge that the refuge is dynamic. That is, past ecological theory has shown that when a prey species is able to retreat from its predator in a fixed refuge space, the basic instabilities of the predator–prey arrangement can be cancelled. But, in the present example, the refuge is effectively a pattern formed by another element in the system , blueberry grow pot the Azteca ant. And the Azteca ant is dynamic in the system, increasing its numbers in proportion to the resources it gains . If the scale insect population increases, there is more food for the ant, and it will therefore make more nests and expand its territory, creating even more refuge area for the scale insect. However, as the ant expands its area of influence , an increasing fraction of the area becomes refuge and, therefore, not available to the adult beetles . At the extreme, there must be some point at which the beetle is unable to find enough prey to continue its population expansion, because almost all of the area would now be a refuge for the scale insect. Therefore, theoretically, the inevitable expansion of the refuge would lead to the eventual local extinction of the beetle predator. It could, of course, be the case that this expected instability of the system does not express itself for diverse reasons or perhaps for an excessively long time. However, purely theoretically, it represents a potential problem for persistence of this control agent.

The theoretical problem is resolved by some very simple natural history observations. A fungal disease, known as the white halo fungus , almost inevitably becomes epizootic , especially when local population densities of the scale insect become large . The fungus can occasionally be found on isolated scale insects, but almost always is most evident when scale insects have built up a significant local population density, and such a buildup can only happen when they are under the protective custody of the Azteca ant.In the end, we see that the Azteca ant plays a key role in the control of this pest. On one hand it protects the scale insect from its adult beetle predator but only in the area of the refuge of the scale, which is defined by the ant itself . On the other hand, it permits the scale insect to build up such large local populations that the white halo fungus frequently becomes epizootic and drives the scale insect to local extinction. It is a curious inverse application of Gause’s traditional competitive exclusion principle, which might be expected to apply between the fungus and the beetle because they share this same food source. It seems unlikely, however, that the scale could be controlled completely by either the beetle or the fungal disease, except in the context of a spatial pattern generated by the Azteca ant . The massive expansion of the ants that might be expected theoretically never happens, partly because of the local effect of the fungal disease and the beetle larvae together reducing the scale insect population locally. Therefore, the dynamic nature of the ant cluster mosaic , always provides a small set of refuges that allows the beetle predator to be maintained throughout the coffee farm. From the point of view of the beetle, it is perhaps ironic that the beetle itself may be involved in the organization of the spatial pattern that is required for its own persistence . There is yet an additional complication. The fungal disease, once it arrives, multiplies extremely rapidly. But, as was noted above, it does not arrive in the first place unless the scale population is large and locally concentrated. Therefore, once the disease gets there, it increases to epidemic levels and wipes out the entire population of scale insects , creating a classical situation of boom and bust and hysteresis in space . Although it is a somewhat complicated argument that has been made in a couple of different ways elsewhere , the disease can clearly generate a locally chaotic dynamic trajectory. Furthermore, as the relevant population gets closer to the ant nest , the oscillations with its disease are expected to be more and more extreme. Eventually, they become so extreme that they transcend the boundaries of a critical value and both scales and disease completely disappear. Note that chaotic trajectories have boundaries , and the equilibrium point at zero is constrained within a basin of attraction.