Tag: hydroponic

Species interactions have received less attention in global change biology than individual species’ responses. In large part, this is because long-term data on species interactions spanning the period of intense anthropogenic environmental change are rare. For example, first flower dates of Japanese cherry blossoms have been recorded in diaries since the ninth century, but we have no equivalent long-term records of cherry tree pollination, leaf microbial communities, or disease incidence. Data describing species interactions are laborious to collect and, in many cases, require technology, such as electron microscopy or DNA sequencing, that was not available until recent years. The lack of long-term data inhibits assessment of how species interactions are impacted by global change and limits our ability to determine how these effects mediate individual species’ distributions, abundances, and ecologies. Variation in species responses to global change has generated concern that interactions which were tightly coupled historically might become decoupled owing to phenological asynchronies. Phenological asynchrony arises when interacting species respond differently to global change—for example, if earlier flowering as a consequence of global warming is not matched by earlier pollinator emergence. Recent meta-analyses have suggested that phenological sensitivity to climate change differs among trophic levels, with lower trophic levels advancing more than higher trophic levels. In one well-documented example, great tit reproduction did not advance in sync with peak food availability for young, leading to potential fitness costs. Similarchanges in interactions between trophic levels may happen as a consequence of other differential responses to global change, such as spatial mismatches between species whose ranges expand poleward or upward in elevation to different extents.

While predictions for phenological and spatial asynchronies are clear, empirical data are sparse, square plant pots and there is still no consensus on whether asynchrony is common or rare, or which traits regulate when asynchronies arise. In the absence of long-term observational data, global change biologists increasingly mine museum collections to investigate how species interactions have shifted over time. Diverse types of data are available only in natural history collections, and they could therefore have wide applicability in global change biology. While natural history specimens are not collected systematically—and their use in ecological and evolutionary research can present challenges—they represent time-series data across much of the globe, span the tree of life, and may be able to fill gaps in species interactions data. Importantly, a large proportion of specimens were collected prior to the intensification of anthropogenic change and therefore may serve as baselines for studying consequences of, for example, invasive species spread, pollution and habitat alteration. Here, we explore the potential for museum specimens to provide insights into interactions between insect herbivores and their host plants. Insects have been eating plants for nearly 400 million years, and these interactions have given rise to much of macroscopic diversity. Herbivores co-evolved with plants, tracking plant speciation or defensive profiles and are frequently specialized. Over the past 12 000 years or so, humans have altered these relationships by domesticating plants and moving them beyond their natural ranges, spraying pesticides, building cities, and changing the global climate. Effects of these global changes on herbivores and their host plants is of critical importance to ecosystem functioning and the provision of ecosystem services. Insect damage—‘herbivory’—drives ecosystem processes, including decomposition and primary productivity. Herbivory also influences ecosystem properties that are of direct importance to people, including food production and tree cover, which are linked to human physical and mental health. In general, warming is expected to increase insect herbivore abundance where insects are living below their thermal optima.

While most insects in temperate and boreal climates probably occupy niches well below their thermal optima—and thus may benefit from warming—warming may cause temperatures to exceed insect thermal optima in areas that are already relatively warm, including the tropics. Insect fitness is not, however, a simple function of mean annual temperature, especially at the local scale. For example, many species have thermoregulatory behaviours that decouple body and air temperatures. In some cases, warming may also have negative effects on insect fitness by reducing snowpack or disrupting diapause. Furthermore, and contrary to the prediction that warming should increase temperate insect herbivore abundance, recent evidence suggests that insect biomass has declined precipitously in Germany since the 1980s. The extent of these declines across space and the mechanism driving them remain unknown, as does whether these declines have occurred in other continents, where comparable long-term insect data were not recorded. While shifts in the global climate will undoubtedly shape species interactions, the local drivers of global change may have complex and nonlinear impacts. Urbanization is a more localized form of global change that is accelerating, with important consequences for plant –insect interactions, and has thus attracted much recent attention in ecology and evolution research. Most people now live in urban areas for the first time in history, and the proportion of the world human population living in urban areas continues to grow. Impacts of urbanization on plant –herbivore interactions could thus have increasing consequence for society via its effects on urban greenery and agriculture. However, the key mechanisms driving insect and plant responses to urbanization remain debated because: urban development, like climate change, has multiple, concurrent effects that are difficult to tease apart experimentally, and we lack long term observational data to determine effects of urbanization over time. Despite these data challenges, studies in the past few decades consistently show that urban development can have profound impacts on plants and insect herbivores. The suite of competing predictions of how insects might respond to global change, and the multitude of mechanisms linking insect herbivore abundance and fitness to herbivory, makes predicting changes in insect damage to plants difficult in the absence of long-term data on historical responses.

Further, herbivory data for assessing competing hypotheses are currently sparse and thus ill-suited for making projections about herbivory change into the future. According to a recent compilation of studies, most short-term herbivory studies include just one year of data , focus on forest plants , are biased toward the northern hemisphere , and record only chewing damage, which represents a fraction of the damage types made by insects. Though short-term studies over space have highlighted that disturbance can have profound effects on plants and herbivores, only few studies focus on disturbed habitats, including cities and farms, areas where plant –herbivore relationships are likely to affect human well being. Here, we propose that biological collections could contribute data that would allow us to test major hypotheses on how diverse plant –herbivore relationships respond to global change. In contrast to observational or experimental studies of insect herbivory, data from museum collections span decades or sometimes centuries, include diverse growth forms and taxa from across the globe, and capture multiple types of herbivore damage. We focus on hypotheses that may be particularly well served by the 380 million vascular pressed plant specimens—herbarium specimens—and the more than 500 million insect herbivore specimens held in museums worldwide that are increasingly available online in the form of images and metadata. In general, we focus on above ground, not below ground herbivory, because intact roots required for assessing below ground herbivory are rarely available on herbarium specimens. Digitization of insect specimens has lagged behind plants owing to the difficulty of capturing three-dimensional specimens and the information from their labels. However, enormous efforts to digitize both plant and insect specimens are underway. Digital collections are also increasingly aggregated in online databases, e.g. the Chinese Virtual Herbarium , LepNet , Symbiota , GBIF , and iDigBio .These databases facilitate ‘big’ data analysis, but are equally as important in helping focus data collection efforts when physical specimens need to be examined. As we discuss below, these specimens can provide a wealth of ecological data that is difficult or impossible to collect using more traditional approaches. In the following sections, we demonstrate how natural history collections may provide unique insights into changing plant –insect herbivore interactions. We focus on species shifts in time and space as a response to recent anthropogenic climate change, and impacts of urbanization, representing one facet of habitat transformation, which is a major driver of current global change. In subsequent sections, we discuss how collections can also reveal species’ rapid adaptive responses to recent global changes, an application that may be particularly consequential for agriculture. Finally, plastic potting pots we review the challenges of natural history collections as sources of long-term data and suggest approaches to some of these challenges, with the goal of removing barriers that have prevented collections from becoming a standard source of data for twenty-first century ecology.Phenology—the seasonal timing of life-history events such as flowering and leaf-out in plants—is both a response to and an indicator of global change. Phenological models, such as the Spring Indices, allow us to map with increasing accuracy the transition from winter to spring across the northern hemisphere. These models integrate daily climate variables from meteorological records to predict day of year of first leaf and first bloom. However, the Spring Indices are calibrated using an extensive network of phenological observations on a single cloned lilac cultivar and two honeysuckle cultivars across the temperate United States .

While meta analyses reveal a consistent fingerprint of climate change on plant phenology, they also reveal large interspecific variation in plant responses. Thus, responses to climate change remain poorly characterized for the majority of plant species. The vast wealth of vouchers within herbaria greatly expand the spatial, taxonomic, and temporal extent of phenological observations and, as a consequence, the inference we can draw across climate space, even for species for which phenology has been documented in long-term observations. Animals are also shifting their phenology with climate change; many species are migrating sooner, advancing seasonal breeding times, and insects are emerging earlier. Natural history collections have been valuable in demonstrating how animal species respond to climate warming. Thanks to the biases of early Victorian naturalists and their attractiveness to contemporary collectors, the Lepidoptera— butterflies and moths, which in their immature stages are herbivorous—have been collected more comprehensively than many other groups and are thus the subject of a large proportion of collections research on animals. Butterfly collections document occurrences of species in time and space and, importantly, the seasonal timing of butterfly flight. Using data from approximately 48 000 collection records of Canadian butterflies, Kharouba et al. were able to show that timing of flight season predictably responded to temperature, and that species with early flight seasons and low dispersal ability appear most sensitive. However, in one recent study, Brooks et al. collected data from 83 500 specimens of British butterflies spanning100 years of climate change which suggested that early flying species might be approaching the limits of their phenological advancement. If advances in butterfly phenology are slowing but their host plants continue to leaf-out and flower earlier, we might observe phenological asynchrony between them. In collections research the potential for phenological synchrony between plants and their pollinators has tended to attract most attention, and collections data have been less frequently used to explore plant–herbivore interactions. However, the few studies that have considered phenological asynchronies between plants and insect herbivores demonstrate the potential for collections to inform such analyses. For example, Kharouba et al. showed that flowering time was more sensitive to temperature than the timing of nectar-feeding butterfly flight, suggesting that caterpillars or adult butterflies of these species might become phenologically mismatched with their host plants if warming continues. While collections data can be extensive for particular taxonomic groups, it is nonetheless rare for collections to capture temporally and spatially matching data on interacting species, such as on both butterflies and their host plants. However, herbarium collections can offer data on phenological sensitivity of plant species and, indirectly, data on their herbivores as captured by the amount of leaf area removed by herbivory. Herbaria may thus offer a unique opportunity to explore how shifts in plant phenology have affected herbivory since the onset of climate change. In figure 2, we outline some possible scenarios describing how warming could affect plant and insect phenology, and how these responses might translate to changes in herbivore damage to plants. For simplicity, we focus on spring phenology and specialized herbivores, though for plants that are commonly eaten primarily by generalists, we could derive an additional set of predictions. Additional factors, such as herbivore developmental plasticity, host plant nutritional quality , relationships with natural enemies, and differential responses among herbivores of a single host plant, might add complexity to the predictions described in figure 2, but could be placed within this general framework.

To compare whether the proportion of occupied nests differed with nest strata or the diversity of nest entrance sizes available, we used generalized linear mixed models with ‘glmer’ in the ‘lme4’ package in R . We compared two models. In the first, we included nest strata , nest size treatment , and the interaction between the two as fixed factors, the vegetation complexity index as a covariate, and site as a random factor. In the second, we removed the VCI. To select the best model, we used the Akaike’s Information Criterion computed with the ‘mass’ package . For both models, we used the binomial error distribution with the logit link. Instead of using the proportion data directly, we used the ‘cbind’ function with number of nests occupied and number of nests that were not occupied as input variables. To examine whether species richness differed with nest strata or the diversity of nest sizes available, we used two methods. First, we compared the mean species richness of ants occupying nests on a plant with GLMMs with ‘glmer’ in the ‘lme4’ package in R . We compared two models. In the first, we included nest strata , nest size treatment , and the interaction between the two as fixed factors, the vegetation complexity index as a covariate, and site as a random factor. In the second, we removed the VCI. To select the best model, we used the Akaike’s Information Criterion computed with the ‘mass’ package . For both models, we used a Poisson error distribution with the log link. Second, we created sample-based species accumulation curves, scaled to the number of individuals, to compare richness in coffee plants vs. trees and diverse vs. uniform nest size treatment plants with EstimateS . We used the number of ant colonies encountered instead of the number of individuals, growing blackberries in containers as ants are social organisms and better captured by number of colonies .

We examined curves for both observed species richness and plotted 95% confidence intervals to statistically compare species richness between treatments. To compare whether community composition of ants differed with strata and with nest size treatment, we used two methods. We used non-metric multidimensional scaling and analysis of similarities in PAST to visually and statistically compare species composition of the ants occupying nests in coffee vs. shade trees and in uniform vs. diverse nest treatments. The ANOSIM compares the mean distance within groups to the mean distance between groups, and can statistically determine separation in species composition between the plots in different treatment groups. For the NMDS and ANOSIM we used the Bray-Curtis similarity index as the similarity measure. Finally, we examined whether common ant species more frequently colonized nests of a certain entrance size or vegetation strata. To compare if nests with certain entrance sizes were more frequently occupied by ants we used an ANOVA followed by a Tukey’s test to compare the mean proportion of nests of each entrance size that were occupied. We only used data from the diverse treatment plants to calculate differences in nest colonization. To compare if certain ant species more frequently occupied certain nest sizes or strata we performed Chi-squared analysis which is recommended for categorical data and tests the likelihood that an observed distribution is due to chance .Ecological studies strive to understand local and regional factors that influence community assembly and species coexistence . Some factors important in for assembly of arboreal twig-nesting ants include presence of a canopy dominant species and resource access through canopy connectivity . Previous studies have found that diversity of nesting resources influences the colonization process of leaf-litter twig-nesting ants and of tropical arboreal ants and that the abundance of nesting resources may impact colonization of arboreal twig-nesting ants .

The study of assembly in ant communities in a spatial context reveals that species sorting, by which different species specialize in a particular habitat, and mass effects, in which species disperse from less to more suitable habitats, are likely important for common and rare species, respectively, in agroecosystems — habitats embedded in landscape mosaics were local communities interact through dispersal . Our study is novel in that we examined colonization in a managed ecosystem looking at two factors and their importance in colonization. In this study, we suggest that nesting resource utilization, specifically different frequencies of occupation of specific nest entrance sizes and specific nesting strata are important drivers of community assembly. Overall, l we found that nesting strata and the diversity of nest entrance sizes did not significantly influence the proportion of occupied artificial nests. Thus, ants use newly available cavities for colonization and nesting resources are somewhat limiting for the community of twig-nesting ants in the habitat studied. In comparison to our study, Powell et al. found that total nest occupancy was higher with higher nest cavity diversity in the Brazilian savanna . It is possible that such distinct results derive from differences in overall nest availability, differences in vegetation and differences in the abundance of particular genera . However, it is important to consider that the near-saturation found in the present study could be a result of adding only one cavity of each size per plant, which could mean that there were not enough nests to be colonized, once the “preferred” sizes were used on every plant —hence not available for other species to occupy. Differences in nest saturation and the proportion of nest occupation between both studies could be due to differences in the number of cavities per size used in the experiment. Thus, it is difficult to say that differences in nest limitation are due to the agroecological context, since previous studies in coffee plantations have found that the community of twig nesting ants are limited by nesting resources , as are ants in natural ecosystems . In addition, differences in occupation dynamics of artificial nests during the colonization phase could potentially change with length of the study. A clear contrast is that the present study lasted three months, a third of the previous study, this difference in time could potentially influence competition for “preferred” cavities during colonization, as these are available for a longer period of time during the colony life cycle. Very little information is available about the reproductive phenology of arboreal twig-nesting ants. The evidence collected from our nests indicates that all common species were producing larvae and pupae, and that most species nests did contain alate males. Two of the common species collected from nests in the present study do experience queen flights during this time period , but information is lacking for the other species. Thus, timing of nest placement may have affected the colonization processes, but it is important to note that many twig-nesting species expand by colony budding, and not only nuptial flights. Changes in the occupation dynamics —i.e. proportion of occupied nests, changes in diversity and species interactions—through time, could be the focus of future studies. Even though diversity of nest entrance sizes did not influence the percentage of occupation overall, frequency of occupation of nests by ants did differ for particular sizes. Higher occupancy was found in middle sizes , these results are similar to Powell et al. in which middle sizes were the most frequently occupied.

The specificity in the use of particular sizes is important in two ways: first, the evolution of ecological specialization underlies the evolution of morphological specialization in ant soldiers, Powell showed that for different species of Cephalotes anincrease in ecological specialization corresponded to a higher head specialization ; in that same study C. persimilis uses cavities that match the size of one soldier’s head and it has also evolved a highly specialized complete headdisc, while less ecologically specialized Cephalotes species, square pot like C. pusilus have evolved a domed-head. Second, such size specialization maximizes individual nest survival and is likely to have a positive effect on overall colony reproduction as shown previously for C. persimilis, which more frequently nests in cavities that fit its head size . On the other hand Cephalotes ants using cavities larger than their soldier’s head, allows them to protect the nest using cooperative blocking . The present study supports the former hypothesis , in that the Cephalotes species present in our study , a domed-headed soldier morphotype, was more frequently found in the largest size , an entrance size much larger than the ant’s head maximum-recorded width . Other Cepahlotes species prefer natural nest sizes between four and up to ten times their head size . If C. basalis shows a similar preference, and if we assume a maximum head size of ~5 mm , than its preferred size might be between the 16 mm2 and 32 mm2 nests offered in this study.Mean species richness was not different in artificial nests on coffee plants and trees, however the diversity of nest entrance sizes increased mean species richness on individual trees and coffee plants. In contrast to a previous study in which diversity of nest cavities did not significantly affect the number of ant species per tree , we did find that providing a diverse array of twig entrance sizes promoted local ant species richness in both coffee shrubs and shade trees. This supports the idea that making a diversity of resources available in both strata supports a more diverse mix of arboreal twig nesting ants. That we found more species richness per tree when providing a higher diversity of nest sizes could indicate that competition for resources might happen more intensively at the local scale, rather than at larger spatial scales. Namely, in a study of leaf-litter twig-nesting ants in shade coffee plantations in Colombia, 80% more species were found when providing a diverse mix of twigs rather than a monospecific collection of twigs showing that diversity of twig-nesting ants is influenced by other aspects of diversity of nesting resources. We found that certain ant species more frequently occupied particular sizes and this may be in part, an explanation for why we found higher species richness on individual plants with a diversity of nest entrance sizes. Armbrecht et al. showed the importance of a diverse mix of twigs for species richness, however the driver in their study was not preference of different ant species for a different species of twigs, but rather an emergent property of a diverse mix of twigs. In our study, we provide evidence that species sorting along a size gradient likely explains the differences observed in mean species richness in uniform vs. diverse treatments. The frequency of occupation differed between sizes for certain ant species, largely following differences in ant head sizes . As small ants can occupy a nest with a wide array of entrance sizes, larger ants can only occupy nests with entrances sizes larger than the workers. Thus providing a wider diversity of nest sizes may allow for greater niche differentiation in the ant community. This outcome might increase the overall richness of the ant community or on individual plants. In our study, larger ants seem to be more size limited than smaller ants, likely because larger ants simply cannot fit into the nests with smaller entrance sizes, and thus are directly constrained by the availability of twigs that fit their body dimensions . In vastly different systems, similar properties operate. For example, in aquatic systems, water temperatures can limit temporal and spatial distribution of certain species as morphological constraints can significantly limit species’ access to suitable habitats . Alternatively, models of exploitative competition have suggested that when two species compete for one limiting resource the result of such competition is determined by the species more capable to attain the lowest equilibrium resource concentration possible, R* . In other words, R* becomes a factor that is the lowest extent to which a certain species can survive in a certain area. Community composition varied between plants with uniform vs. diverse nest entrance sizes, as well as in coffee plants and shade trees. Our results are consistent with previous studies that have investigated ant stratification in the rainforest, where there is a strong partitioning of ant species in the leaf litter, lower vegetation and canopy . Likewise, tropical ant activity is often higher in the canopy than in the litter environment, and species composition differs between the canopy and litter assemblages .

The total run time per sample was 61.67min. Electron ionization was performed with a source temperature of 230o C and the quadrupole at 150o C. The wine samples were measured using synchronous scan and selected ion monitoring . The mass spectrometer scanned from m/z 40 to 300. Compounds were detected using between two and six selected ions. Data was analyzed using MassHunter Qualitative Analysis software . After normalization with 2-undecanone internal standard, results were expressed as peak areas. Compounds were tentatively identified in the mass spectrometry spectrum of the peaks and confirmed by comparison to the National Institute of Standards and Technology database . The ions used SIM for each compound and retention times were reported previously by . The odor activity value thresholds were obtained from a selected review of published literature of young red wines and were used in comparing the monitored compounds . Meteorological data collection and climactic conditions at the experimental site for the 2020 and 2021 growing seasons are described in detail by Marigliano et al. . Briefly, there were 1762.7°C growing degree days accumulated in 2020 compared to 1572.3°C GDDs accumulated in 2021, with similar GDD accumulation from April to June in both years. Compared to the 10-year average , the 2020 growing season accumulated more growing degree days by 1 October. The 2021 growing season was a cooler year with less accumulated GDD than the 10-year average. The total precipitation at the experimental site from 1 March 2020 to 30 September 2020 was 84.1mm, a notable 100.5mm less precipitation than the 10-year average for the experimental site. Drought conditions continued into the 2021 water year, growing blueberries in containers with 66.9 mm of precipitation between 1 March 2021 and 30 September 2021.

Precipitation only occurred in March and April 2021 and was negligible in the following months. Given the severe drought conditions in both experimental years, precipitation had a negligible effect on plant water status in control and shaded treatments with 25% ETc replacement, as demonstrated by no significant effects on stem water potential integrals between control and shaded treatments in either experimental year .Grapes resulting from field treatments were vinified under the same conditions in both years. In 2020 alcohol content was the highest in D1 and D4 wines , while alcohol content and residual sugar concentration was lowest in C0 in 2020. All shade film wines contained more alcohol and residual sugar than C0. In 2021, alcohol content and residual sugar concentration was unaffected across all wines. In 2020, pH was only decreased in D3 wines. In 2021, C0 wines had the lowest pH compared wines from shaded grapes. Among the shaded treatments, D4 and D5 wines had higher pH compared to D1 and D3 wines. In 2020, titratable acidity only increased in D3 wines compared to C0, D1 and D5 wines. C0, D1, D4 and D5 wines were indistinguishable in titratable acidity. While C0 had one of the lowest values for TA in 2020, C0 wines in 2021 had one of the highest TA values, along with D3 and D5 wines. The lowest TA value was observed in D4 wines from 2021. Color intensity within the 2020 wines varied considerably, with the D4 having the greatest value for CI . In 2021, D4 again had the highest values for CI, while the remaining wines were statistically not different from each other. Hue decreased only in D3 wines during the 2020 vintage, while there was no effect of shade films of wine hue during the 2021 vintage . The trend for the percentage of polymeric anthocyanins was consistent in both vintages. D1 and D4 had the highest percentage of polymeric anthocyanins, while D5, D3and C0 wines had less . In 2020, D1 and D4 wines had higher TPI values compared to C0 and D3 wines. In 2021, TPI of wines was not affected by shade films except for D4. 3.3 Wine flavonoid content and profile Wine anthocyanin profiles were separated into glucosides, 3-acetylated and coumarylated anthocyanins .

The total free anthocyanin concentration was the lowest in C0 wines compared to shade film treatments in 2020. Concentrations of 3-glucosides and 3-acteylated glucosides increased for all anthocyanins under shading treatments compared to C0, except for peonidin 3-acetyl-glucoside and cyanidin 3-glucoside in which shading treatments had no effect. The composition of coumarylated 3’4’5’-hydroxylated anthocyanin modifications was largely impacted by shading, with the largest concentrations detected in C0, D1 and D5 wines. Overall, the ratio of di- to tri-hydroxylated anthocyanins was the largest in C0 wines and the lowest in D5. Conversely in 2021, total free anthocyanin concentrations were the highest in D4, C0, and D1 wines. Anthocyanin modifications due to shading treatments were more varied in 2021 compared to 2020. Overall, wines from D4 had the most 3-glucosides and 3-acetylated glucosides, while C0 and D5 consistently had less. Coumarylated anthocyanin concentrations were reduced in D3 and D5 wines compared to C0 wines. This was not consistent with the concentrations observed in 2020. Likewise, there was no statistically significant effect on the anthocyanin hydroxylation ratio in 2021 wines, while shading had an impact on anthocyanin hydroxylation in wines in 2020. Nine flavonol compounds were monitored in wines using HPLC . For all monitored flavonol compounds except myricetin-3-glucuronide, C0 wines consistently had the highest concentrations in 2020 compared to shaded wines, with D4 and D5 wines following in flavonol concentration. Subsequently, C0 also had the highest wine flavonol concentration when calculated as total flavonols in 2020. A similar trend occurred in 2021. C0 wines from 2021 also contained greater concentrations of each flavonol compared to shaded treatments, as well as total flavonol concentration.The wine aroma profiles from the 2020 and 2021 vintages were analyzed with and 29 volatile compounds were identified and categorized into their respective compound classes . The aromas profiles of wines depended highly on vintage, resulting in distinct aroma profiles.

Generally, in 2020, total higher alcohols were unaffected by shade treatments, except for isoamyl alcohol and benzyl alcohol. Wines produced from shaded fruit had similar concentrations of isoamyl alcohol while the C0 had the lowest isoamyl alcohol concentration. Benzyl alcohol concentrations were reduced in D3 and D5 wines compared to C0, D1 and D4 wines. In 2021, shading treatments did not impact the concentration of higher alcohols in the resulting wines except for benzyl alcohol, which increased in 2021 D3 wines compared to all other treatments. Acetate esters and fatty acid ethyl esters showed varied effects in wines due to shading in 2020. C0 and D5 had the lowest ethyl acetate concentrations compared to the other shade treatments. Likewise, isoamyl acetate was reduced in C0, D4 and D5 wines compared to D1 and D3 wines. Among the shade film treatments , ethyl hexanoate and ethyl octanoate concentrations were comparable between D1 and D5 wines and were greater than concentrations found in D3 wines. C0 and D5 wines were indistinguishable in ethyl butyrate, ethyl-2-methylbutyrate and ethyl valerate in 2020, with D1 and D3 wines having the highest concentrations of each these ester compounds. Isobutyric acid increased in D4 in 2020. In 2021, there were no significant impacts of shading on acetate esters, fatty acid ethyl esters, ethyl butyrate, ethyl-2-methylbutyrate or ethyl valerate. The effect of shade films on various terpenes and norisoprenoids was highly dependent on vintage conditions. Alpha-terpinene was highest in D5 wines but was significantly reduced in D1 and D3 wines in 2020. The D4 wines had the most cis-rose-oxide while C0 wines had the least. Linalool concentrations were reduced in C0, D4 and D5 wines. Among the shaded treatments, nerol concentrations were enhanced in D5 wines in 2020, square pots while there was no effect of shading on nerol concentration in 2021. D5 did not differ from the C0 in nerol concentration in 2020. Farnesol in D3 was reduced in 2020 whereas farnesol concentrations were not affected in 2021 wines. Conversely, nerolidol was unaffected by shade films in 2020, whereas significant decreases in nerolidol concentrations were observed in D4 and D5 wines in 2021. It was observed that β-damascenone were elevated in 2020 in C0 wines, yet differences in β- damascenone concentrations were non-significant between shade film treatments. In 2021, only significant differences in β-damascenone concentrations were observed in wines, with C0 wines containing the most β-damascenone and D5 wines containing the least. β-ionone concentrations were not statistically significant between all treatments in 2020 and 2021. To determine the effects of partial solar shading on wine chemistry, flavonoid composition, and aromatic profiles of wines we conducted a principal components analysis for both vintages . In 2020, PCA indicated that PC1 accounted for 30.8%, and PC2 accounted for 22.1% of the total variance. The C0 treatments clustered together, separately from the partial solar shading treatments. The separation along PC1 was explained by the ratio of di- to tri-hydroxylated anthocyanins in wines, norisoprenoids and flavonols, as well as lower CI, alcohol content and TPI. The separation along PC2 was explained by TA, pH, terpenes and the percentage of polymeric anthocyanins in wine samples. In 2021, PCA indicated PC1 accounted for 29.9%, and PC2 accounted for 22.2% of the total variance. The C0 treatments again separated from shade film treatments, but less so than in 2020. The separation in PC1 was again explained by the ratio of di- to tri-hydroxylated anthocyanins, along with the total glucosides, total methylated anthocyanins, and total anthocyanins. The separation of C0 was along PC2 and thus was associated with higher concentrations of flavonols, terpenes, norisoprenoids, and polymeric anthocyanins in wine. We analyzed the relationships further between the variables monitored with a correlation analysis in wines . In 2020, CI in wines had the strongest positive correlation with TPI and acids .

Alcohol percentage and ketones were also positively correlated to TPI and acids, although less so than CI. Ketones also were very strongly positively correlated with higher alcohols, while higher alcohols were less strongly correlated to acids. Conversely, flavonols were strongly negatively correlated with acetate esters and other esters in wines. Norisporenoids and pH were less negatively correlated to acetate esters. Fatty acid ethyl esters particularly showed to be negatively correlated with TA. In 2021, the strongest positive correlations in wines were between total anthocyanins and total glucosides and total methylated anthocyanins . Total coumarylated anthocyanins were significantly and positively correlated to total anthocyanins, methylated anthocyanins, and total glucosides. Strong negative correlations were found between hue and ester compounds including fatty acid ethyl esters and acetate esters. A strong negative correlation existed between the ration of di- to tri-hydroxylated anthocyanins and total acetylated anthocyanins. Lastly, total higher alcohols and pH were strongly negatively correlated with each other. In hot viticulture regions, there is a desire to reduce excessive alcohol content in wines due to marketability and taxation concerns. Numerous studies have demonstrated that partial solar radiation exclusion is an effective method for reducing the amount of ethanol in wines by reducing TSS in shaded clusters . However, in the present study, C0 wines consistently had the lowest alcohol content and the lowest concentration of residual sugars in 2020 compared to shaded fruit, despite grapes at harvest having similar TSS values across the treatments . This may be due to the composition of sugars in the grape berry being affected by excessive cluster temperatures in C0 fruit. Sepúlveda and Kliewer showed that heat stress at 40°C post-veraison decreases glucose and fructose in the grape berry. During heat wave events post-veraison, cluster temperatures in C0 reached a maximum temperature of 58°C, exceeding the point at which glucose and fructose content is altered . Additionally, the production of non-fermentable sugars such as arabinose, raffinose and xylose are known to be present in the grape berry . Genes involved in the production of these sugars have been shown to be upregulated under heat stress conditions in grapevine . While the grape berry is 95-99% glucose and fructose at harvest, these non-fermentable sugars are included in the metric of total soluble solids . As a result, while TSS was unaffected by shade films , the proportion of fermentable to nonfermentable sugars may be impacted, thus leading to 2020 C0 wines with reduced alcohol content. This difference in alcohol content between 2021 wines was not observed most likely due to the 2021 growing season being cooler with less GDDs than 2020 .

Currently, Sangiovese is also the cultivar with the highest number of clones registered in the Italian National Catalogue of Grapevine Cultivars showing 130 clones in 2020. Despite its global distribution and importance, there are a limited number of international studies on Sangiovese grapes and wines. The most substantial scientific works were presented at theInternational Symposium of Sangiovese between 2000 and 2004 in Italy. Some of these studies showed the influence of regionality and terroir on the flavor profile of Sangiovese wine. Recent research has focused on the relationship between Sangiovese clones’ grape quality and their oenological properties and polyphenol composition of Sangiovese wines and the mouthfeel perception. A first study about the chemical characterization and comparison of Sangiovese wines from California and Italy was recently reported. The findings demonstrated that the California Sangiovese wines have common characteristics with the Italian wines, particularly related to some volatile grape-derived compounds. These components can be associated with the varietal character of Sangiovese in both regions as the wines in each region retain a common set of characteristics, particularly for the varietal volatiles that originated from grape. Typical characterization of wines includes relationship between sensory profile and volatile compounds, polyphenols, and elements. A much smaller number of studies have compared the chemical and sensory profiles of wines from multiple countries, including Malbec from Argentina and California, red wines from Australia and China, and Sauvignon blanc wines from France, New Zealand, Spain, South Africa, and the United States. Regional typicality is an important concept for the wine industry as it not only delineates geographic areas but also comprises wines with recognizable sensory characteristics and composition. Characterizing regional differences in wines requires studying the intrinsic quality as its inherent physical-chemical characteristics. Moreover, perceived quality is rated by experts, critics, and consumers, gallon pot which altogether defines a profile that describes common regional characteristics among wines. The concept of perceived quality is even more critical when the studies are related to typical wine such as a Protect Designation of Origin . In fact, in a PDO context or when describing a wine belonging to a particular region and produced with a recognizable grape variety, the intrinsic quality of wines could be summarized by the typicality assessment.

Typicality is defined as the characteristics of a product from a terroir, meaning that the product is representative of its terroir, with terroir defined by two dimensions such as the environmental factors and the variety, cultivation, and wine making practices. Among them, the effect of interactions between the natural environment and the vegetal material is known to be a major driver of wine typicality and quality. Thus, typicality can be defined as a set of properties of belonging and distinction. Considering the absence of defects as a pre-requisite, some authors proposed that the intrinsic quality is the resultant of three different profiles: an eligibility profile, whose parameters are common to all wines ; an identity profile , whose parameters are related to the grape variety and the terroir; a style profile related to the brand, expression of the kind of wine making. The eligibility profile can change over time without affecting the identity of the wine, while the identity profile cannot change because it represents the distinct characteristics that define the typicality of a wine. Finally, the style profile can chance overtime as function of the market or the winery brand needs, without altering the identity profile of the wine. It is important to highlight that even in countries with consolidated protected designation of origin and protected geographical indication systems, a scientific approach of typicality still represents a challenge in terms of concept and sensory methodologies. Since Sangiovese is one of the most wide-spread Italian red grapes and a very terroir-linked variety, it is important to study this grape cultivar on a broader scale in order to answer some key questions such as the relationship between the chemical differences, in terms of eligibility and identity profiles, of the wines from different countries and the sensory profiles of the relevant wines,and how do Tuscan experts perceive the typicality of the Sangiovese wines from California and Italy, and which sensory descriptors best describe the wines. The purpose of this study was to evaluate the intrinsic and perceived quality of Sangiovese wines from Italy and California, defining both similarities among wines from the same region of origin and the main differences between wines from Italy and California.

Twenty commercial wines from the 2017 harvest were collected to be representative of both regions . The selection of the wines was made according to the study of the 2016 vintage by the same authors. All of the wines used in this study were sourced from commercial producers, were required to be 100% Sangiovese, and were not oak barrel aged. All wines selected did not show off-flavors. A minimum of 6 bottles were received for each wine sample. As the wines were made solely from Sangiovese grapes under commercial wine making conditions, the differences in composition should reflect the regional styles. In California, the wines were chosen from the following American Viticultural Areas : Central Coast, North Coast and Inland Valley region. The AVAs incorporate the following counties/regions: Amador County, Napa Valley, Santa Ynez Valley, Paso Robles, Saint Joaquin valley and Alameda County. In Italy, the wines were chosen from Tuscany representing the wine areas of Chianti Classico, Chianti, and Montalcino. As all wines used in this study were commercial products; there was no control over the viticultural or wine making practices, and all participating wineries were asked for a “best representation of Sangiovese. The eligibility chemical characteristics were represented by standard parameters, color indices, and polyphenol composition; the identity chemical characteristics were represented by the volatile fractions of the wines. The style requirement was represented by the chemical variables related to wine aging . The standard parameters were measured with FT-IR analyses according to OIV/OENO Resolution 390/2010 and carried out by means of a FOSS WineScan . Color intensity and hue were measured according to the method of Glories and the total phenols index as described by Ribereau-Gayon. The ultraviolet-visible absorbance of the samples was measured on an Agilent spectrophotometer Cary 8454 UV–visible diode array detector, and the software used was UV–Visible ChemStation . Milli-Q water was used as a reference . The polyphenol contents of the wines was determined according to Girardello et al. methods using an Agilent 1260 Infinity HPLC equipped with an autosampler, temperature-controlled column compartment, and a diode array detector.

The polyphenol composition of the wines is shown in Table S1 . Free volatile compounds were determined according to the method developed previously by HS-SPME GCMS. The analytical system for the determination was a Gerstel MPS2 autosampler mounted to an Agilent 6890N gas chromatograph paired with an Agilent 5975 mass-selective detector constituted the analytical system for the GC-MS analysis. The software used was MSD ChemStation . The volatile composition of the wines is shown in Table S2 . All the wine samples were analyzed in triplicates for all chemical parameters.The descriptive sensory analysis panel took place in the J. Lohr Sensory Room at the Department of Viticulture and Enology, University of California Davis. The panel consisted of 11 judges. We recruited the participants from students, staff, and friends of UC Davis based on availability and interest. The protocol was exempt by the internal regulatory board . The panel leader trained the panelists in six 60-min training sessions. In the first sessions, the panelists were presented with a range of Sangiovese wines and invited to describe them, generating descriptors and idea of standards. In the subsequent sessions, gallon nursery pot the judges were provided of subset of samples and reference standards until they reached the consensus about the attributes and the score sheet sequencing. The references were prepared from food and household products commonly available in the supermarket. The level of training of the panelists was checked by an individual evaluation of a subset of the samples and the analysis of the data. The eligibility sensory profile was described by the following 6 attributes: Sour, Sweet, Bitter, Viscous, Astringent, and Hot/Burning. The identity profile was defined by the following 13 aromatic attributes: Citrus, Floral, Black pepper, Dried fruit, Barnyard, Dark Fruit, Rubber, Cherry, Alcohol, Honey, Red berries, Earthy, and Bell pepper in-mouth flavor. One attribute described the style requirement, namely Oak odor and Oak in-mouth flavor. The wine samples were served at room temperature in black glasses covered with plastic lids. Each sample contained a constant volume of 40 mL of wine. Wines were tasted blind, coded with randomized three-digit numbers, and served twice during the training period. At the end of the training period, the panel had consensus on 14 aromas, 3 taste and 3 mouthfeel descriptors for the wines . The panelists had the opportunity to familiarize themselves with the data entry system FIZZ network for collecting the attribute ratings. Before each evaluation, panelists took an aroma quiz on the consent aromas, and on the first data collection day, panelists tasted the taste and mouthfeel standards. The experimental design consisted of 12 wines served across judges in a balanced block design in triplicate. We labeled the wine samples with different 3-digit codes for each panelist. Panelists evaluated all wines in a total of 6 sessions, where one session consisted of 6 samples. Panelists rated the wines in individual, ventilated, and light isolating tasting booths under white light. For attribute ratings, we used an unstructured 15-cm line scale anchored by the wording “not present” to “very intense”, except for viscous, where the wines were rated from “thin” to “thick”. Judges were required to spit all the wine samples and wait 30 s between samples to clean their palates with water and unsalted crackers. At the end of each session, panelists had snacks, and after the study, panelists received a gift card. All the training and evaluation sessions were performed in about two months .Eleven Italian experts , working with Sangiovese wine, with extensive experience with the different expression of Sangiovese wine produced in most important wine producing Italian areas, were involved in a sensory evaluation session in which three tests were performed: Napping® test and the typicality and color assessments. The same 12 wine samples submitted to the descriptive analysis were evaluated except for the sample 12C that was damaged during shipment from California to Italy. These sensory evaluations took place in the wine sensory laboratory at the Department DAGRI, University of Florence . The expert panelists were informed at the beginning of the study that the wines were all Sangiovese of the 2017 vintage. They were then instructed to assess the color of the samples presented under a white light, in glasses covered with plastic lids, labeled with capital letters, from A to M. The panelists were instructed as follows: “Imagine that you wanted to explain to someone what a Sangiovese wine color is. To explain, you can suggest to this person to evaluate a wine. For each wine presented, you must answer the following question: Do you think that this wine is a good example or a bad example of what a Sangiovese wine color is?” The score of each sample was assigned on a categorical scale, from 1 to 7, anchored at left to “very bad color” and on the right to “excellent color”. After this, judges proceeded to the Napping and typicality assessments. The Napping is a specific variant of Projective Mapping, a method originally proposed for applied sensory studies by Risvik et al. to describe overall differences among samples. In both the Napping and typicality sessions, panelists were presented with the 11 wine samples plus one replicate. The evaluations were performed in isolated, ventilated sensory booths under red lights, to eliminate bias attributed to color differences. Each wine sample consisted of 25 mL of wine at room temperature presented, in clear 190 mL standard tasting glasses covered with plastic lids, and labeled with different random three-digit codes for each panelist and session. For both tests , a complete randomized and balanced experimental design was followed for the presentation order. In the Napping session, the 12 samples were simultaneously presented to the panelists, who were then required to project them on a two-dimensional space , in a way that reflected their perceived sample differences, i.e., by placing samples perceived as similar close to each other, and samples perceived to be more different further apart.

To gain statistical evidence of miRNA differential expression driven by the environment and/or genotype, we made pairwise comparisons, keeping constant the developmental stage, and evaluating the miRNA modulation among vineyards or between cultivars . The analyses reveal that some miRNAs are differentially expressed between the two genotypes grown in the same environment, but also that a number of miRNAs are modulated by the environment. In particular the number of differentially expressed miRNAs is higher in ripened berries , while no miRNAs are differentially expressed at bunch closure stage . In details, 14 reads are differentially expressed at pea size stage, in at least one comparison, corresponding to 6 distinct miRNA families; 27 reads are modulated at 19 ◦Brix stage, corresponding to 12 miRNA families and 35 reads are differentially expressed in berries at harvest, corresponding to 12 miRNA families. It is worth noting that 4 of the 6 families modulated in the berries at pea size, are still present among the miRNAs differentially expressed in the berries sampled at 19 ◦Brix and at harvest , even though not always in the same comparisons. Some of the modulated miRNAs, both novel and known are intriguingly connected to berry development and secondary metabolism, even though most of the modulated families are still uncharacterized, or with targets not clearly involved in berry ripening and development, and deserve further studies to fully understand their biological roles. Using high throughput sequencing coupled with robust bioinformatics pipelines we analyzed small RNAs derived from the berries of Cabernet Sauvignon and Sangiovese, grown sideby-side in three vineyards, representative of different grapevine cultivation areas in Italy . The size distribution profiles of our libraries were in general consistent with previous reports in berry grapevine, black plastic pots for plants where the 21-nt class was more abundant than the 24-nt class .

Our analysis revealed dynamic features of the regulatory network mediated by miRNAs and other small RNAs, at the basis of genotype-environment interactions. Plants evolved a series of pathways that generate small RNAs of different sizes with dedicated functions . Although the various small RNA classes have been intensively studied, we are still far from understanding how many small RNA pathways exist, and how they are connected . Additionally, new classes of small non-coding RNAs continue to be discovered and many studies demonstrate a substantial redundancy and cross-talk between known small RNA pathways . Estimating the exact percentage of the plant genome covered by small RNA-generating loci still remains a challenge. By applying static cluster analysis, we investigated small RNA abundances across the genome, identifying 4408 small RNAs producing hotspots. We analyzed their expression in different cultivars, environments and developmental stages, highlighting that the majority of the considered small RNA producing regions was modulated in different conditions. This suggests a strong influence of small RNAs in the response to environment in grapevine berries. Only 462 small RNA-generating loci, corresponding to about 10% of the total, were expressed in all the analyzed libraries, possibly involved in essential biological pathways. Comparing the two cultivars, we observed, with few exceptions, that Cabernet Sauvignon berries have a higher number of expressed sRNA-generating loci than Sangiovese berries when collected in the same conditions . Considering the fact that small RNAs are implicated in the regulation of gene expression in several processes , the higher number of small RNAs expressed in Cabernet Sauvignon compared to Sangiovese berries may reflect a buffering effect of small RNAs influencing grapevine response to diverse growing environments. We believe that these characteristics may have contributed to the wide diffusion of Cabernet Sauvignon, allowing its wide cultivation in almost all wine producing countries. This is not the case for Sangiovese whose cultivation is more restricted.

It is worth noting that Sangiovese is considered a very unsettled grapevine cultivar , showing a wide range of variability in response to year, clone and bunch exposure . Differently, Cabernet Sauvignon is a cultivars showing less inter-annual differences in terms, for example, of concentration of secondary metabolites . To better evaluate varietal differences in response to the environment, we calculated the CS/SG ratio for the small RNA producing hotspots in the three vineyards. An interesting example is found in green berries sampled in Riccione. A region on chromosome 4 showed a 390-fold change in the small RNA abundance, when comparing Cabernet vs. Sangiovese . Most of the reads produced in this region are 21 nt long and are also phased in intervals of 21 nt from both strands, typical of a phased locus . The gene in this locus, also known as VvRD22g, encodes a BURP domaincontaining protein, involved in an ABA-mediated abiotic stress response, which persists still after long periods of stress . The small RNAs profile suggests that the locus is regulated by phased siRNAs similarly to the mechanisms already described for PPR, NB-LRR, and MYB gene families . This is a clear example of GxE interactions since the BURP domain gene modulates phased siRNAs production in the two cultivars only when grown in Riccione. When removing the threshold of minimum cluster abundance set to 5 HNA, in the CS/SG ratio, a high number of clusters with fold change greater than 50 was found, where one of the libraries has 0 HNA and the other any number greater than 30 HNA. This fact suggests a very strong modulation of the expression of small RNAs between the two cultivars, which is more or less pronounced depending on the vineyard where the berries were cultivated. A similar situation was observed comparing the expression level of small RNAs between reciprocal hybrids of Solanum lycopersicum and S. pimpinellifolium . The ripening process of grapevine berries is highly affected by the environment and we observed the impact of the environment on the ripening process in the expression of small RNAs.

The most relevant observation is that Riccione is very peculiar in relation to the activation of sRNA hotspots, as indicated by the high number of Riccionespecific clusters and by the extreme modification it induces in the CS/SG ratio : in Riccione in fact this ratio decreases in green berries and increases in ripened berries, and this is not observed in any other vineyard; in addition to this the already discussed example of BURP domain gene, is observed in Riccione, as well. Riccione is the most diverse environment when compared to Montalcino and Bolgheri. Riccione is located at the Adriatic coast and has a temperate sub-littoral climate, while Montalcino and Bolgheri are both located in Tuscany with typically Mediterranean climate. Moreover, both cultivars show a peculiar profile of small RNA loci during berries ripening, in Riccione. The expression of small RNA loci in Cabernet Sauvignon berries drastically changed during development, especially when collected in Riccione , not only in the number of active loci but also in the different genic or intergenic disposition: ripened berries have a 2.6-fold increase in small RNA loci active in genic regions. Differently, when Sangiovese is grown in Riccione, there is a very high number of small RNA loci active in green berries, mainly associated to transposable elements that remains almost stable during development although the proportion of intergenic loci is reduced. Sangiovese berries collected in Montalcino show a 2.5- fold increase of small RNA producing loci during development. Differences during berry development between the cultivars may explain their different behavior in different environments, and the characteristics of each vineyard may favor one or other variety according to their demands. For example, Sangiovese needs a long growing season with sufficient warmth to fully ripen . Consequently, drainage pot cooler environments will require a reprograming of Sangiovese gene expression in order to achieve ripening. Other factors such as composition of soil, level of humidity, photoperiod and density of cultivation may be exerting the same influence on the ripening of the berries triggering the activation of different small RNA loci. Applying a conservative pipeline to the analysis of our 48 small RNA libraries, we recognized 89 known and annotated grapevine miRNAs. In addition, when compared to previous reports in grapevine we identified 7 completely novel miRNAs plus 26 homologous to other plant species, but novel to grapevine. This is a remarkable number considering the stringency of our pipeline and that our study is based only on four developmental stages of berries. The outline of miRNA accumulation across samples is different from that of sRNA-producing loci. While the expression of sRNA-generating regions allows distinguishing very well between ripened and green berries and also between cultivars , the accumulation of miRNAs shows a clear distinction only between ripened and green berries, and when the berries were green, we observe a further dichotomy separating the two cultivars and the two green developmental stages. The same pattern of miRNA accumulation among green and ripened berries of grapevine was observed when we described the miRNA expression atlas of Vitis vinifera . Comparing the distribution of miRNAs expressed throughout our samples, we found a set of 39 miRNAs ubiquitous or nearly ubiquitous to all the libraries, and very few miRNAs specific of a cultivar, vineyard or developmental stage.

All these 39 miRNAs belong to known vvi-miRNA families. With few exceptions, the same set of miRNAs was also found expressed in all the small RNA libraries constructed with different tissues of the grapevine cv. Corvina , where the population of expressed miRNAs appears highly variable apart from a well-defined group of miRNAs, probably related to the basal metabolism. These findings are also consistent with previous report in grapevine where a small number of known tissue-specific miRNAs was described . Considering the ripening process as shown in the heat maps , and the correlation dendrogram, it is clear that most miRNAs are modulated during the developmental process. For some miRNA families, we observed the same peculiar patterns of miRNA accumulation, previously described in the grapevine miRNA atlas , e.g., an increase of accumulation toward ripening for miR156 f/g/i, and a decrease for miR166c/e, miR172d, miR319, and miR396a/b, but this is not the main focus of our paper. To establish genotype and environmental influence on miRNA modulation, we performed a statistical analysis that revealed a number of miRNAs differentially expressed. Being aware of the fact that we had only two biological replicates, we applied the exact test as implemented in the EdgeR package. This test has been recently judged a very robust tool that can be used in experiments similar to our, because of its low false positive rate and relative high true positive ratein the presence of a fold change higher than 4 . Considering berries at the same developmental stages, we compared Sangiovese vs. Cabernet Sauvignon in a given vineyard and Montalcino vs. Bolgheri, Montalcino vs. Riccione, and Bolgheri vs. Riccione keeping the cultivar fixed. In total we performed 9 pairwise comparisons for each developmental stage. In general, we observed that berries at 19 ◦Brix and at harvest show a higher number of differentially expressed miRNAs. The most interesting examples are represented by two novel miRNAs, whose predicted targets are related to the biosynthesis and accumulation of secondary metabolites, which are of crucial importance in grapevine berries, since its quality depends mainly on its metabolites . The candidate grapem1191 is differentially expressed in Sangiovese between Riccione and Bolgheri and was predicted to target the transparent-testa 12 gene that encodes a multidrug secondary transporter-like protein involved in the vacuolar accumulation of the flavonoid proanthocyanidin in different species including grapevine . Also, in grapevine some studies provide evidences that the intracellular transport of acylated anthocyanins is catalyzed by a MATE transporter . The grape-m1355 seems to be involved in four different pathways, all related to secondary metabolites. It is differentially expressed in Montalcino between the two varieties and was predicted to target a cinnamoyl reductase-like protein , which is part of the of the polyphenol biosynthetic pathway ; a cinnamyl alcohol dehydrogenase involved in the lignin biosynthesis ; a phenylacetaldehyde reductase , which catalyzes, in tomato, the last step in the synthesis of the volatile 2-phenylethanol, important for the aroma and flavor of many foods ; and different bifunctional dihydroflavonol 4-reductases . DFR catalyzes the first step in the conversion of dihydroflavonols to anthocyanins and are responsible for the production of colored anthocyanins .

Water use efficiency predictions are further complicated by the results of combination studies of elevated temperature, reduced soil water availability, and elevated CO2, which reveal synergistic effects. In an open top chamber study, combining temperature and CO2 did not result in gs being significantly reduced, contrary to results of elevated CO2 alone . When latent heat is trapped, overheating subsequently decreases the activity of RUBISCO activase, for most plants at temperatures higher than 37°C , and in grapevine between 35-40°C, varying by species . The elevated CO2 and temperature treatments showed an increase in transpiration , and the effects of drought were only temporarily delayed . Temperature and elevated CO2 had an additive effect on plant leaf area for multiple grapevine clones , highlighting that overall higher leaf area without increased WUE could be detrimental for heat stressed vines. Measurements of predawn water potential were more negative in vines at elevated CO2, indicating the demand for soil water availability of vines with increased productivity . Notedly, the production of fine roots was positively impacted by an elevated CO2 treatment, which would theoretically increase water absorption of water available . There remain inconsistent predictions of the effects of elevated CO2 on grapevine whole plant water use efficiency, which seem to be contingent upon other factors such as soil water availability, temperature, and variety of grapevine. With the evidence from these studies of elevated CO2 and combination studies of soil water availability and temperature, grapevines most likely will not benefit from a long-term increase in photosynthesis under elevated CO2. The lack of soil water available and biological temperature thresholds for RUBISCO will limit the gains in photosynthesis, pot with drainage holes and more likely the vines will struggle to release latent heat as temperatures rise.

Grapevine phenology is categorized into four life cycle stages of periodic development: budburst, flowering, veraison, and maturation. The grapevine phenological cycle is a two-year process; bud formation occurs in the first year which develop into shoots in the second year. Therefore, clusters are significantly impacted by the previous year’s climate . For grapevine grown at elevated CO2, advances in phenology compound significantly over seasons . This is likely the result of stored carbon photosynthate from the productive previous year. As a result, it can take several years to observe the effects of elevated CO2 on grapevine phenology , which leads to the question of: “To what extent does elevated CO2 impact the timing of phenological stages over the long-term?” Studies of Arabidopsis, another C3 flowering plant, provide insight to the mechanisms of phenological changes observed in grapevine. Excess carbohydrates may act similarly to phytohormones to delay the upregulation of genes involved in flowering time, as well as cell wall invertases in the meristem that down regulate photosynthesis under treatments of elevated CO2, which leads to earlier flowering . For grapevine, it is possible that excess photosynthate could trigger early flowering through the transfer of carbohydrates from leaves. One of the most robust findings to support this hypothesis is that growth under elevated CO2 results in increased carbohydrate reserves in plants . The sugars produced by photosynthesis contribute only a fraction of the source of carbon needed for rapid growth and development from budbreak to flowering and sugar accumulation in berries at veraison, the remaining needed for these growth spurts is mobilized from long-term storage of total nonstructural carbohydrates in trunks and roots . Over several growing seasons, storage of carbohydrates in the trunk will be impacted by elevated CO2 and could therefore contribute to shifts in phenology.

In a greenhouse study of fruiting cuttings where sugar accumulation in berries was measured, elevated CO2 increased the rate of ripening correlated with the photosynthetic rate . The effect of elevated CO2 on phenology was greater than the treatment of temperature elevated by 4°C . Therefore, an increase in total nonstructural carbohydrates could be a driver of advances in phenology long term, on its own, as well as with concomitant increases in growing season temperatures. Carbohydrate reserves regulate the growth and differentiation of flowers, which only occurs after the grapevine shoot is resource independent from the rest of the vine . These findings suggest that with an increase in carbon reserves stored as starch in roots, trunks and canes, second season shoots may grow faster and achieve independence earlier in the growing season. This could contribute to early flowering as a result of lifted competition for resources between vegetative and reproductive growth. In contrast, long-term studies in grapevine decreasing the leaf to fruit ratios decreased essential reserves of the TNC in the roots . The well-known viticultural technique of strategic leaf removal has been shown to delay maturation, highlighting the importance of carbon availability for phenological development . While the mechanism for phenological shifts in grapevine grown under elevated CO2 is under-studied, these shifts have been quantified using FACE experiments. The combination of elevated CO2 and temperature in OTC caused an advance in flowering time by three days and veraison by two weeks . The impact of elevated CO2 on phenological timing is greatest during the period between fruit set to veraison and this impact increases when combined with a temperature treatment . During fruit set, elevated CO2 treatments with and without temperature treatments increased total soluble solids , as well as decreased anthocyanins and malic acid concentration, which would contribute to an earlier veraison and harvest . However, the impact of high temperature may have a greater impact on this phenological period .

Grapevines vulnerable to frost damage will suffer from early budburst, with subsequent losses in yield . One consequence of increased shoot vigor at elevated CO2 is the expected increase in bud fertility, which will likely increase the number of flowers per vine . Changes in cluster density and phenological timing impact the carefully articulated annual harvest. Unbalanced sugar/acid ratios resulting from early harvest decrease the quality of grapes and wine produced, discussed further in the “Berry and Wine Chemistry” section below . Shifting the lifecycle of grapevine will have a global impact on winegrape production.Fruit composition is a major area of concern for growers and winemakers alike, especially aromatic compounds. The changes in pest interactions, physiology, and timing of veraison in response to elevated CO2 will collectively impact the resulting grape and wine quality . For successful wines, in the grape berry there is a balance of acid and sugar at harvest. Increasing atmospheric carbon available impacts the balance as ripeningadvances and sugar accumulation is accelerated . Flavonoids and anthocyanins are important for the flavor, color, and mouthfeel of wine. The molecular analysis from the original Italian FACE experiments showed increases in total flavonoids, total anthocyanins, and total non-anthocyanin flavonoids in the wine produced with carbon enriched grapes grown at 700 mg/L , which typically would affect the color and mouthfeel of wine. Interestingly, a subsequent experiment using 500 mg/L CO2 open top chambers determined there were significant increases in ethyl 2-methylbutyrate , isoamyl acetate , ethyl hexanoate , ethyl octanoate , butyric acid , and isovaleric acid concentrations and a significant decrease in ethyl acetate concentration in wines produced from enriched CO2 grapes after one year , which contribute to the balance of floral and fruity characteristics in wines . In the second year they found lower methionol , 1-octanol , and 4- ethylguaiacol , and they found higher ethyl lactate and linalool concentrations, large pot with drainage although these changes in berry chemistry did not appear to significantly affect the quality of wine produced . These results agree with early studies led by Bindi et al. that did not find significant effects on the quality of wine produced from grapes grown at elevated CO2 . Although the changes observed in compounds contributing to flavor have been noted as so far insignificant for quality, a major concern for winemakers is the increase in alcohol content resulting from an increase in sugar concentrations in berries, as a result of higher CO2 concentrations . In the past, winemakers have added sugar to the fermentation to increase the final alcohol percentage , depending on legal regulations for winemaking.However, in recent years winemakers have begun removing sugar through processes like reverse osmosis in order to prevent alcohol levels from rising . Overall, elevated CO2 is altering the balance of sugar accumulation, the levels tartaric and malic acids in berries and wine, and the impact on wine quality continues to be investigated . The most recent FACE studies on grapes continue to evaluate the berry chemistry and quality developing over years of exposure to elevated CO2. The VineyardFACE in Germany analyzed must from grapes after pressing and did not find a significant increase in sugar content from conditions of carbon enrichment . The Gonçalves et al. study also concluded that changes in water availability and heat stress could change their predictions in wine quality.

We should expect that with the shifts in phenology and physiological changes to berries, early harvest will impact the quality of grapes in terms of reaching maturation too quickly . Viticulturists could also anticipate altered physiological demands to have long-term impacts on berry quality . In contrast to the ecological pressures discussed above, the rates of some fungal infections may be reduced in elevated CO2 scenarios. With higher carbon allocation to roots, grapevine mycorrhizal colonization may be promoted by elevated CO2 , which has been shown to protect grapevine against the nematode Xiphinema index by stimulating defense gene response . A study of elevated CO2 on several varieties of grapevine seedlings showed a reduced severity of the infection of Xanthomonas campestris pvviticola, a vector of bacterial canker in immature grapevine . This may be the result of lower stomatal conductance ; with stomatal aperture reduced, there is less opportunity for bacteria to invade the leaf pores . Also, researchers recorded a reduced instance and severity of powdery mildew infection in cv Barbera, at elevated CO2 . The Geisenheim VineyardFACE site recorded changes in the bunch architecture but did not see an increase in the frequency of B. cinera, botrytis bunch rot, a necrotrophic fungus, occurrence . Changes in leaf chemistry phenotype, specifically carbon content, , will increase the pressure of grapevine pests in future climates. Increasing available carbon dioxide, without a concomitant increase in nutrient levels in the soil, leads to an increase in C:N ratios in leaves . Insects consume at higher rates when nitrogen has been diluted to meet their nitrogen intake needs and chewing insect pests will generally eat more leaf tissue in elevated carbon dioxide scenarios . Elevated CO2 increased individual survival rates and increased the fecundity of female mealybugs, which eat phloem of grapevine damaging the temporal and perennial plant tissue . The European grapevine moth, Lobesia botrana, is a major problem for European vineyards, affecting both the berries and flowers of grapevines; and has already invaded North and South American vineyards . L. botrana is also responsible for spreading Ochratoxin A-producing Aspergillus fungi, which typically spikes in occurrence during hotter and drier years . At higher temperatures simulating future climate conditions, L. botrana female growth rate and pupal mass increased , while researchers found a down regulation of expression of ethyleneresponsive factors, which suggests grapevines can become more vulnerable to herbivory or abiotic stress under future climate change as these are the major stress and defense response factors . A comprehensive study of soil and elevated CO2 showed the decomposition pathway is altered by the carbon-, nitrogen-, and phosphorus-acquiring enzymes in the soil with a significant increase in nematode density . More than 4,000 plant-parasitic nematodes exist, posing a well-known global issue for grapevine, reducing total crop production by 8.8- 14.6%, and one of the worst threats from the nematode Xiphinema index is GLRV . Under elevated CO2 conditions, if ethylene is suppressed and salicylic acid is increased, it is likely that grapevine will struggle with an increase in pest and disease vectors such as nematodes and fungi . Grapevines largely rely on human intervention for defense against pests and diseases , and this reliance could increase in future climates. Consider the grapevine “immune system” as weakened in terms of chemical defense, but some altered carbon dynamics under elevated CO2 may be beneficial for reducing severity of pest pressure.

The very concept is embedded in the colonial relationship itself, implying as it does that there is some land that is not sacred. Moreover, the federal government did not allow “sacred land” as a legal land-claim defense until the 1970s, and its documentation must meet standards defined by the federal government, a bizarre requirement since the federal courts seldom demonstrate much wisdom in cosmological or religious matters. Thus Indian spiritual connections to the environment are frequently examined and negotiated in light of contemporary residential development, land management activities, and outdoor recreation as well as legislative activities and the outcomes of litigation. Spiritual connections are seldom straightforward however. Stephen Jett suggested that there may be varying degrees of sanctity for Navajo, although he is unclear about whether such a continuum emanates from his own analysis of the situation or from Navajo interpretations. In addition, James Griffith explained in detail the multi-valent possibilities of places where ideas of sacredness held by the Tohono O’Odham, Yaqui, Mexican, and Anglo American peoples intersect. Also, Linea Sundstrom attempted to set the record straight for the Black Hills using archival documents in an ethnohistorical reconstruction identifying what is held sacred in the area, since when, and by whom, including at least seven tribes. The secular popularity of the subject of sacred lands has not come without repercussions. People are dehumanized and cultural complexity trivialized when non-Indian environmentalists furate on indigenous ecological spirituality and activity while ignoring other significant aspects of community and culture. Bruce Willems-Braun makes this point about marginalization of the Nuuchah-nulth of British Columbia, square pot who often have been denied an active role in debates over wilderness preservation because a perspective on the environment has frequently been imposed on them rather than asserted by them.

Three decades ago Brian Goodey called on geographers to work as researcheradvocates in support of Indian tribes and their economic development. Sincethat time, land use, economic development, and tourism, especially related to gambling, have undergone rapid change in Indian Country. When coordinated with tribal interests, research into these areas generally has been viewed positively by tribal governments. There are a number of geographers and their colleagues who are working for tribes directly or as consultants, or are pursuing research on these topics independently. Data problems are a major concern among these researchers, especially difficulties encountered in using census and Bureau of Indian Affairs or Department of Indian Affairs and Northern Development , the Canadian equivalent of the BIA, data for analysis of population and economic and community development. A critical issue in tribal planning is the need to establish a framework for government-to-government relations between tribal and city, county, and state governments when work must be accomplished in a regional context. A major applied research effort on this topic was carried out and summarized by Shirley Solomon, a geographer working with the Northwest Renewable Resource Center in Seattle. This collaboration produced extensive coordination of tribal groups and local governments. One study found that access to health services was severely restricted on the Round Valley Reservation in northern California not only because of its extreme distance from comprehensive medical facilities, but also because trained medical and dental professionals could not be consistently retained. Another found that Montana Indian women were twice as likely as non-Indians to have to travel outside the county to a birthing facility and even more likely to have poor obstetric care anywhere near where they live.

None of this will come as a surprise to readers familiar with rural life, especially life in Indian Country. But geographers are novices to health care planning and are only just beginning to realize their expertise may be of use in examining aspects of health care such as location, access, travel distance, and sense of place. The new multicultural conceptions of health and well-being now beginning to replace standard medical-geography models undoubtedly will encourage more interest in Indian health issues. Federal legislation in the 1980s and 1990s made gambling a potentially significant component of reservation economic development, the impact of which was examined in two edited books and several articles by geographers. The case studies indicate rapid growth in gambling accompanied by some economic benefit accruing to most participating tribes. Long-term stability, increasing competition from other gambling facilities, subsequent social polarization, and the politics of wealth redistribution both inside and outside reservation boundaries are recognized as problems. Regional studies for Connecticut, the Dakotas, New Mexico, and Oklahoma also are included in these publications. The sale of American Indian arts and crafts and the associated appeal of cultural tourism have long played a role in Indian economic life, although the benefits have been distributed unevenly from region to region. In the past, such activities were generally directed and controlled by non-Indians, and the bulk of revenues and proceeds went to non-Indians. While there are some exceptions, this pattern often continues today. Several geographers have studied the consequences of tourism for Native populations and the increased effort to gain local control of tourism facilities and activities. Geographers also have begun applying postcolonial or other social theory to American Indian studies. Generally anglophonic postcolonial studies have emerged from and focus on the British colonial experience; accordingly, postcolonial American Indian research is well represented within Canadian universities, most notably the University of British Columbia. These researchers seek to deconstruct and assess the imprint of a European worldview on the lands, minds, and bodies of Indians. They draw inspiration from postcolonial literature and French intellectuals Henri Lefebvre, Jacques Lacan, Jacques Derrida, and Michel Foucault.

These diverse theoretical currents are brought together in studies of colonial surveillance and control of Indian spaces, the appropriation of such spaces through ethnocentric cartographic or other textual representation, and the various Indian responses to such practices. Nicholas Blomley has employed similar themes, investigating Indian blockades in British Columbia as a mechanism for tribal reappropriation of space, and Robert Galois has attempted a revised empirical overview of historical Kwakiutl settlement free from earlier colonial biases. Explaining the mechanisms of group representation and identity is fundamental in several studies: Berry’s comparative work on waterscapes and representation for Paiute and non-Paiute communities in northern Nevada; Dennis Crow’s inquiry about reservations as “low places”; Deur on the Makah whale hunt revival; Galois on the period of colonial consolidation in British Columbia; Peters’ examination of the apparent conflict between “city person” and “Indian” identities in Canadian cities; and Silvern’s study of treaty-rights conflicts in Wisconsin. Also, the spatial aspects of gender are influential in the way places and people function. Karen Morin examined British women’s constructions of Indians as Others in historic “contact zones” like the railroad depots of the American West, Peters explained the role of Indian females in subverting urban spaces in Canada, and Wishart considered the role and status of women in Pawnee society, and the general place of Indian women as subjects of geographical research. In an unusual link to demographic study, Robert Jackson sifted the evidence of population decline in the US Southwest and developed the thesis that deaths were related not only to disease, but also to place/space destruction and reductions in cohesion, identity, and sense of place. In a similar vein, R. Douglas K. Herman provided insight into changes in Hawai‘ian identity in response to colonization and corresponding shifts in environmental knowledge and language. The question of whether there exists a pan-Indian sensibility about place and space also has been of some interest. In a 1976 interview, N. Scott Momaday proposed and described what he termed a pan-Indian ecological sensibility bound into Indian identity, an ethos distinguished from those of non-Indian North Americans by the presence of what he termed a sense of “reciprocal appropriation.” Then geographers David Stea and Ben Wisner wrote of a panIndian ecological worldview projected outward in solidarity with other indigenous or Fourth World peoples. Since then a few geographers have examined pan-Indianness as an evolving identity crucial to understanding the link between place and action, drainage collection pot and one has written about the men’s movement’s appropriation and wild distortion of elements of this identity.56 Others have shown how Indian identities are materialized in places as disparate as battlefield and massacre sites, cultivated gardens, migration corridors, and interior landscapes of the mind. The Inuit have been of particular interest, as shown in studies attempting to explain their mental landscape representations or “mental maps,” their navigational skills, and their secondary position to environmentalists’ identification with stranded whales and stripped seals. Place names or toponyms are also of longstanding interest to geographers, and another intersection where the work of anthropologists has been influential. Most recently the intimate links among social structure, individual and group identity, and place were illustrated by geographers studying Chinook Jargon, Inupiat, Hawai‘ian, Navajo, and Inuktitut place names. The study of Indian and Inuit maps accelerated during the past ten years and helped revitalize cartography as a discipline with an historically and culturally situated subject matter, and not merely a narrow technical one. A group of writers have examined the interpretations made of Indian or Inuit maps by nineteenth- and twentieth-century explorers and government agents, and the discourses into which these documents entered, including those of contemporary scholarship.60 Map exchanges between Indians or Inuit and Europeans or Euro-North Americans occurred everywhere in North America, but extant examples and thus much of the research effort is concentrated in the continent’s interior plains from Texas to Saskatchewan, and in the Arctic.

These maps are seen as emanating from different assumptions and discourses about the world as a home for humans and non-human others when compared to what Europeans and Euro-North Americans were producing. Indian maps actively disrupted European discourse on geography, history, and identity, and represent one side of what was and still is to some extent an unbridgeable gap in worldviews. The other side, the European and Euro-North American mapping of Indian Country, has also come under scrutiny. This cartography spans the last five hundred years, but geographers have tended to study comparatively recent examples. They have questioned these maps, often finding them ideological weapons serving non-Indian interests rather than the simple and innocent mimetic representations that the general public and their makers imagined them to be. Several others deserve attention because they emanate from Indians or Inuit themselves and represent a new trend of “mapping back” or “counter-mapping” the colonizers. A Zuni Atlas was published because Zuni elders, in their effort to press a land claim, decided it was time to make certain protected geographical information available to the public for the first time. In part this atlas is notable because it reveals some Zuni sacred sites in order to demonstrate land occupancy beyond present reservation boundaries. But it does so using maps drawn at a scale too small for the reader to navigate. Thus the sites were revealed for litigation purposes, but still kept safe from unwanted visitors. The Inuit have been especially active in counter-mapping. First, they created a map series containing their own place names inscribed on Canada’s official topographic maps, then they assembled the Nunavut Atlas, an extensive compilation of wildlife patterns and other environmental data gathered from Inuit elders. These may be viewed both as attempts to overcome disruption of the traditional oral transfer of geographic information from one generation to the next and also as nationalistic outpourings in anticipation of the creation of the new Territory of Nunavut in 1999. Robert Rundstrom has taken note of these developments and their implications in an article and map review. GIS are a completely new technological development within the past two decades but are understood as a natural extension into the digital era of gathering and mapping geographical data. To put it most broadly, GIS are computer systems for the gathering, storage, and manipulation of geographical coordinates and associated statistical data about anything on the earth’s surface, expressions of which can be made in either paper or digital form. They are more than computerized mapping systems because they can transform information in ways uncommon or unknown in traditional map making and they easily link with orbiting satellites and the remotely sensed digital images that satellites transmit. In the past five years, a national American Indian GIS association has developed and is closely linked to the leading corporate manufacturer of GJS software; tribes, therefore, have been very active participants in GIS applications and research. Continued efforts to link the US Census Bureau’s Topologically Integrated Geographic Encoding and Referencing file maps with tribal information has also helped some tribes initiate detailed spatial data analysis of the lands and people administered by tribal government.

The measurement was done using AC current excitations of 0.1 – 20 nA at 0.5 – 5.55 Hz using a DL 1211 current preamplifier, SR560 voltage preamplifier, and SR830 and SR860 lock-in amplifiers. Gate voltages and DC currents are applied, and amplified voltages recorded, with a home built data acquisition system based on AD5760 and AD7734 chips.In crystalline solids, orbital magnetization arises from the Berry curvature of the bands and intrinsic angular momentum of the Bloch electron wave packet. Although the orbital magnetization often contributes—at times substantially—to the net magnetization of ferromagnets, all known ferromagnetism involves partial or full polarization of the electron spin. Theoretically, however, ferromagnetism can also arise through the spontaneous polarization of orbital magnetization without involvement of the electron spin. Recently, hysteretic transport consistent with ferromagnetic order has been observed in heterostructures composed of graphene and hexagonal boron nitride, neither of which are intrinsically magnetic materials. Notably, spin-orbit coupling is thought to be vanishingly small in these systems, effectively precluding a spin-based mechanism. To host purely orbital ferromagnetic order, a system must have a time reversal symmetric electronic degree of freedom separate from the electron spin as well as strong electron-electron interactions. Both are present in graphene heterostructures, where the valley degree of freedom provides degenerate electron species related by time reversal symmetry and a moir´e superlattice can be used to engineer strong interactions. In these materials, a long wavelength moir´e pattern, arising from interlayer coupling between mismatched lattices, vertical towers for strawberries modulates the underlying electronic structure and leads to the emergence of superlattice minibands within a reduced Brillouin zone.

The small Brillouin zone means that low electron densities are sufficient to dope the 2D system to full filling or depletion of the superlattice bands, which can be achieved using experimentally realizable electric fields. For appropriately chosen constituent materials and interlayer rotational alignment, the lowest energy bands can have bandwidths considerably smaller than the native scale of electronelectron interactions, EC ≈ e 2/λM, where λM is the moir´e period and e is the electron’s charge. The dominance of interactions typically manifests experimentally through the appearance of ‘correlated insulators’ at integer electron or hole filling of the moir´e unit cell[13, 19], consistent with interaction-induced breaking of one or more of the spin, valley, or lattice symmetries. Orbital magnets are thought to constitute a subset of these states, in which exchange interactions favor a particular order that breaks time-reversal symmetry by causing the system to polarize into one or more valley projected bands. Remarkably, the large Berry curvature endows the valley projected bands with a finite Chern number, so that valley polarization naturally leads to a quantized anomalous Hall effect at integer band filling. To date, quantum anomalous Hall effects have been observed at band fillings ν = 1 and ν = 3 in various graphene heterostructures, where ν = An corresponds to the number of electrons per unit cell area A with n the carrier density. Although orbital magnetism is generally expected theoretically in twisted bilayer graphene, no direct experimental probes of magnetism have been reported because of the relative scarcity of magnetic samples, their small size, and the low expected magnetization density they are predicted to have.In the absence of significant magnetic disorder ferromagnetic domain walls minimize surface tension. In two dimensions, domain walls are pinned geometrically in devices of finite size with convex internal geometry. As discussed in Fig. 5.15, we observe pinning of domain walls at positions that do not correspond to minimal length internal chords of our device geometry–suggesting that magnetic order couples to structural disorder directly.

This is corroborated by the fact that the observed domain reversals associated with the Barkhausen jumps are consistent over repeated thermal cycles between cryogenic and room temperature. Together, these findings suggest a close analogy to polycrystalline spin ferromagnets, which host ferromagnetic domain walls that are strongly pinned to crystalline grain boundaries ; indeed, these crystalline grains are responsible for Barkhausen noise as it was originally described. Although crystalline defects on the atomic scale are unlikely in tBLG thanks to the high quality of the constituent graphene and hBN layers, the thermodynamic instability of magic angle twisted bilayer graphene makes it highly susceptible to inhomogeneity at scales larger than the moir´e period, as shown in prior spatially resolved studies. For example, the twist angle between the layers as well as their registry to the underlying hBN substrate may all vary spatially, providing potential pinning sites. Moir´e disorder may thus be analogous to crystalline disorder in conventional ferromagnets, which gives rise to Barkhausen noise as it was originally described. A subtler issue raised by our data is the density dependence of magnetic pinning; as shown in Fig. 5.3, Bc does not simply track 1/m across the entire density range, in particular failing to collapse with the rise in m in the Chern magnet gap. This suggests nontrivial dependence of either the pinning potential or the magnetocrystalline anisotropy energy on the realized many body state. Understanding the pinning dynamics is critical for stabilizing magnetism in tBLG and the growing class of related orbital magnets, which includes both moir´e systems as well as more traditional crystalline systems such as rhombohedral graphite. In order to understand the microscopic mechanism behind magnetic grain boundaries in the Chern magnet phase in tBLG/hBN, we used nanoSQUID magnetometry to map the local moir´e superlattice unit cell area, and thus the local twist angle, in this device, using techniques discussed in the literature. This technique involves applying a large magnetic field to the tBLG/hBN device and then using the chiral edge state magnetization of the Landau levels produced by the gap between the moir´e band and the dispersive bands to extract the electron density at which full filling of the moir´e superlattice band occurs .

The strength of this Landau level’s magnetization can be mapped in real space , and the density at which maximum magnetization occurs can be processed into a local twist angle as a function of position . It was noted in that the moir´e superlattice twist angle distribution in tBLG is characterized by slow long length scale variations interspersed with thin wrinkles, across which the local twist angle changes rapidly. These are also present in the sample imaged here . The magnetic grain boundaries we extracted by observing the domain dynamics of the Chern magnet appear to correspond to a subset of these moir´e superlattice wrinkles. It may thus be the case that these wrinkles serve a function in moir´e superlattice magnetism analogous to that of crystalline grain boundaries in more traditional transition metal magnets, pinning magnetic domain walls to structural disorder and producing Barkhausen noise in measurements of macroscopic properties. In tBLG, a set of moir´e subbands is created through rotational misalignment of a pair of identical graphene monolayers. In twisted monolayer-bilayer graphene a set of moir´e subbands is created through rotational misalignment of a graphene monolayer and a graphene bilayer. These systems both support Chern magnets. Both systems are also members of a class of moir´e superlattices known as homobilayers; in these systems, the 2D crystals forming the moir´e superlattice share the same lattice constant, and the moir´e superlattice appears as a result of rotational misalignment, as illustrated in Fig. 5.17A. Homobilayers have many desirable properties; the most important one is that the twist angle can easily be used as a variational parameter for minimizing the bandwidth of the moir´e subbands, container vertical farming producing the so-called ‘magic angle’ tBLG and tMBG systems. Homobilayers do, however, have some undesirable properties. Although local variations in electron density are negligible in these devices, the local filling factor of the moir´e superlattice varies with the moir´e unit cell area, and thus with the relative twist angle. The tBLG moir´e superlattice is shown for two different twist angles in 5.17B-C across the magic angle regime; it is clear that the unit cell area couples strongly to twist angle in this regime, illustrating the sensitivity of these devices to twist angle disorder. The relative twist angle of the two crystals in moir´e superlattice devices is never uniform. Imaging studies have clearly shown that local twist angle variations provide the dominant source of disorder in tBLG . It is hard to exaggerate the significance of this problem to the study of moir´e superlattices. Phenomena discovered in tBLG devices are notoriously difficult to replicate. Orbital magnetism at B = 0 has only been realized in a handful of tBLG devices, and quantization of the anomalous Hall resistance has only been demonstrated in a single tBLG device, in spite of years of sustained effort by several research groups. A mixture of careful device design limiting the active area of devices and the use of local probes has allowed researchers to make many important discoveries while sidestepping the twist angle disorder issue- indeed, some exotic phases are known in tBLG only from a single device, or even from individual scanning probe experiments- but if the field is ever to realize sophisticated devices incorporating these exotic electronic ground states the problem needs to be addressed.There is another way to make a moir´e superlattice. Two different 2D crystals with different lattice constants will form a moir´e superlattice without a relative twist angle; these systems are known as heterobilayers . These systems do not have ‘magic angles’ in the same sense that tBLG and tMBG do, and as a result there is no meaningful sense in which they are flat band systems, but interactions are so strong that they form interaction-driven phases at commensurate filling of the moir´e superlattice anyway. Indeed, many of the interaction-driven insulators these systems support survive to temperatures well above 100 K.

The most important way in which heterobilayers differ from homobilayers, however, is in their insensitivity to twist angle disorder. In the small angle regime, the moir´e unit cell area of a heterobilayer is almost completely independent of twist angle, as illustrated in 5.17E-F. A new intrinsic Chern magnet was discovered in one of these systems, a heterobilayer moir´e superlattice formed through alignment of MoTe2 and WSe2 monolayers. The researchers who discovered this phase measured a well-quantized QAH effect in electronic transport in several devices, demonstrating much better repeatability than was observed in tBLG. The unit cell area as a function of twist angle is plotted for three moir´e superlattices that support Chern insulators in 5.17G, with the magic angle regime highlighted for the homobilayers, demonstrating greatly diminished sensitivity of unit cell area to local twist angle in the heterobilayer AB-MoTe2/WSe2. MoTe2/WSe2 does have its own sources of disorder, but it is now clear that the insensitivity of this system to twist angle disorder has solved the replication issue for Chern magnets in moir´e superlattices. Dozens of MoTe2/WSe2 devices showing well-quantized QAH effects have now been fabricated, and these devices are all considerably larger and more uniform than the singular tBLG device that was shown to support a QAH effect, and was discussed in the previous chapters. The existence of reliable, high-yield fabrication processes for repeatably realizing uniform intrinsic Chern magnets is an important development, and this has opened the door to a wide variety of devices and measurements that would not have been feasible in tBLG/hBN. The basic physics of this electronic phase differs markedly from the systems we have so far discussed, and we will start our discussion of MoTe2/WSe2 by comparing and contrasting it with graphene moir´e superlattices. In tBLG/hBN and its cousins, valley and spin degeneracy and the absence of significant spin-orbit coupling combine to make the moir´e subbands fourfold degenerate. When inversion symmetry is broken the resulting valley subbands can have finite Chern numbers, so that when the system forms a valley ferromagnet a Chern magnet naturally appears. Spin order may be present but is not necessary to realize the Chern magnet; it need not have any meaningful relationship with the valley order, since spin-orbit coupling is absent. MoTe2/WSe2 has strong spin-orbit coupling, and as a result, the spin order is locked to the valley degree of freedom. This manifests most obviously as a reduction of the degeneracy of the moir´e subbands; these are twofold degenerate in MoTe2/WSe2 and all other TMD-based moir´e superlattices.

The techniques described above still have some limitations, and chief among them is a limited range of electronic densities that they can reach. Of course, the gold standard of electron density modulation is the ability to completely fill or deplete an electronic band, which requires about one electron per unit cell in the lattice. Chemical doping can achieve enormous offsets in charge density, sometimes as high as one electron per unit cell. This limitation isn’t fundamental and there are some ideas in the community for ways to improve it, but for now it remains true that electrostatic gates can modify electron densities only slightly relative to the total electron densities of real two dimensional crystals. As it stands, electrostatic gating can only substantially modify the properties of a crystal if the crystal happens to have large variations in the number and nature of available quantum states near charge neutrality. For many crystals this is not the case; thankfully it is for graphene, and for a wide variety of synthetic crystals we will discuss shortly. Electrostatic gating of two dimensional crystals was rapidly becoming a mature technology by the time I started my PhD. So where does nanoSQUID magnetometry fit into all of this? A variety of other techniques exist for microscopic imaging of magnetic fields; the most capable of these other technologies recently developed the sensitivity and spatial resolution necessary to image stray magnetic fields from a fully polarized two dimensional magnet, plastic pots 30 liters with a magnetization of about one electron spin per crystalline unit cell, and this was widely viewed within the community as a remarkable achievement.

We will shortly be discussing several ferromagnets composed entirely of electrons we have added to a two dimensional crystal using electrostatic gates. Because of the afore- mentioned limitations of electrostatic gating as a technology, this necessarily means that these will be extremely low density magnets with vanishingly small magnetizations, at least 100 times smaller than those produced by a fully polarized two dimensional magnet like the one in the reference above. It is difficult to summarize performance metrics for magnetometers, especially those used for microscopy. Many magnetometers are sensitive to magnetic flux, not field, so very high magnetic field sensitivities are achievable by simply sampling a large region, but of course that is not a useful option when imaging microscopic magnetic systems. Suffice to say that nanoSQUID sensors, which had been invented in 2010 and integrated into a scanning probe microscope by their inventors by 2012, combine high spatial resolution with very high magnetic field sensitivity. This combination of performance metrics was and remains unique in its ability to probe the minute magnetic fields associated with gate-tunable electronic phenomena at the length scales demanded by the size of the devices. Gate-tunable phenomena in exfoliated heterostructures and nanoSQUID microscopy were uniquely well-matched to each other, and although at the time I started my graduate research only a small handful of gate-tunable magnetic phenomena had so far been discovered in exfoliated two dimensional crystals, nanoSQUID microscopy seemed like the perfect tool for investigating them. This makes SQUIDs excellent magnetic field sensors, with the caveat that they do not sample the magnetic field at a point, but averaged over a region A. Making a SQUID is as easy as depositing a superconducting material onto a surface in the correct shape, and it can be done using many of the same techniques used to produce other microscopic electronic devices, like photolithography and thermal evaporation.

This is sufficient for many applications, but it presents some issues for producing sensors for scanning probe microscopy. Scanning probe microscopy is a technique through which any sensor can be used to generate images; we simply move the sensor to every point in a grid, perform a measurement, and use those measurements to populate the pixels of a two dimensional array . This can of course be done with a SQUID, and many researchers have used SQUIDs fabricated this way to great effect. But the spatial resolution of a scanning SQUID magnetometry microscope is set by the size of the SQUID, and there are limits to how small SQUIDs can be fabricated using photolithography. It is also challenging to fashion these SQUIDs into probes that can be safely brought close to a surface for scanning; photolithography produces SQUIDs on large, flat silicon substrates, and these must subsequently be cut out and ground down into a sharp cantilever with the SQUID on the apex in order to get the SQUID close enough to a surface for microscopy. In summary, the ideal SQUID sensor for microscopy would be one that was smaller than could be achieved using traditional photolithography and located precisely on the apex of a sharp needle to facilitate scanning. As is so often the case when developing new technologies, we have to make the best of the tools other clever people have already developed. In the case of nanoSQUID microscopy, the inventors of the technique took advantage of a lot of legwork done by biologists. Long ago, glass blowers found that hollow glass tubes could be heated close to their melting point and drawn out into long cones without crushing their hollow interiors. Chemists used this fact to make pipettes for manipulating small volumes of liquid, and biologists later used the techniques they developed to fashion microscopic hypodermic needles that could be used to inject chemicals into and monitor the chemical environment inside individual cells in a process called patch-clamping. A rich array of tools exist for producing these structures, called micropipettes, for chemists and biologists. Eli Zeldov noticed that these structures already had the perfect geometry to serve as substrates for tiny SQUIDs. By depositing superconducting materials onto these substrates from a few different directions, one can produce superconducting contacts and a tiny torus of superconductor on the apex of the micropipette. The same group of researchers successfully integrated these sensors into a scanning probe microscope at cryogenic temperatures.

The sizes of these SQUIDs are limited only by how small a micropipette can be made, and since the invention of the technique SQUIDs as small as 30 nm have been realized. We call these sensors nanoSQUIDs, or nanoSQUID-on-tip sensors. A few representative examples of nanoSQUID sensors are shown in Fig. 1.4. A characterization of the electronic transport properties of such a sensor, and in particular the sensor’s response to an applied magnetic field, is shown in Fig. 1.5. NanoSQUID microscopes share many of the core competencies of more traditional, planar scan-ning SQUID microscopes. They dissipate little power, and the measurements they generate are quantitative and can be easily calibrated by measuring the period of the SQUID’s electronic response to an applied magnetic field. Measuring a magnetic field with a SQUID does not require optical access; many other magnetic field measurement techniques do. Together, these facts mean that scanning SQUIDs are often the best tools available for probing extremely low temperature phenomena. NanoSQUID sensors also have many advantages over planar SQUIDs. The most obvious, of course, has already been discussed, and that is their higher spatial resolution. A less obvious advantage- indeed, an advantage that became clear only after the first nanoSQUID sensors were fabricated and tested- is the geometry of the thin superconducting contacts, which under normal circumstances are aligned with the axis of the applied magnetic field. Large magnetic fields tend to destroy superconducting phases, so superconducting devices are all limited by the maximum magnetic fields at which they can operate. This so-called critical field HC is not an intensive property; there is a large-size limit that can be measured and tabulated for different materials, but the critical field of an individual piece of superconductor is a strong function of geometry. A thin superconducting film in the plane of an applied magnetic field can accommodate much higher magnetic field magnitudes than can be accomodated by a large piece of the same superconductor. The bulk limit for lead at low temperature is about 80 mT; we routinely make lead nanoSQUIDs that can survive magnetic fields of 1 T, round plastic pots and we have on occasion made nanoSQUIDs that can survive magnetic fields above 2 T. It turns out that many of the most useful magnetic imaging techniques are limited to low field operation. This thesis will focus primarily on low field phenomena, but there are also many magnetic phenomena that require high magnetic fields to appear, including the quantum Hall effect and a variety of magnetic phase transitions. The nanoSQUID technique is useful for studying these as well. NanoSQUID sensors have some unique disadvantages as well. Like planar SQUIDs, nanoSQUIDs require superconductivity to function, which limits them to fairly low operating temperatures. In planar SQUIDs it is often possible to keep the SQUID itself cold while scanning over a much hotter sample, but nanoSQUID sensors are extremely poorly thermalized to their scan heads, which means that they generally are thermalized either to the surface over which they are scanning or to the black body spectrum of the vessel in which they are contained . This gives nanoSQUID sensors some interesting capabilities, namely that under the right conditions they can function as extremely sensitive scanning probe thermometers, but it also comes with some drawbacks. NanoSQUIDs composed of superconductors with critical temperatures below 4.2 K, the boiling point of helium-4 at atmospheric pressure, must thus have actively cooled thermal radiation shields to operate in very high vacuum, and of course imaging of hot samples is completely out of the question for these sensors.

A variety of exciting opportunities exist for the application of sensitive magnetic imaging techniques to biological systems, and this is not a realistic option for nanoSQUID sensors. NanoSQUIDs are quite fragile and can be easily destroyed by vibrations, necessitating vibration isolation systems, and the superconducting film on the apex of the micropipette is quite thin, typically between 15 and 20 nm, so superconducting materials that oxidize in air will be quickly degraded. Thankfully indium and lead do not oxidize rapidly, but they do oxidize at a finite rate, so nanoSQUIDs composed of these materials only last for a few days when left in air. Storage in high vacuum can improve their lifespan, but generally not indefinitely. In summary, scanning probe microscopes fitted with nanoSQUID sensors can function as magnetometry microscopes with 30-250 nm resolution. They are capable of operating at very low temperatures and magnetic fields of up to several Tesla. Their high sensitivities allow them to detect the minute magnetic fields emitted by electronic phases composed entirely of electrons forced into a two dimensional heterostructure with an electrostatic gate. We will discuss some of the properties of two dimensional heterostructures next. Many crystalline compounds have cleavage planes; that is, planes along which cracks propagate most readily. When such compounds are stressed beyond their yield strength, they tend to break up into pieces with characteristic shapes that inherit the anisotropy of the chemical bonds forming the crystal out of which they are composed. Indeed, this observation was a compelling piece of early evidence for the existence of crystallinity, and even atoms themselves. There exists a class of materials withcovalent bonds between unit cells in a two dimensional plane and much weaker van der Waals bonds in the out-of-plane direction, producing extraordinarily strong chemical bond anisotropy. In these materials, known as ‘van der Waals’ or ‘two dimensional’ materials, this anisotropy produces cleavage planes that tend to break bulk crystals up into two dimensional planar pieces. Exfoliation is the process of preparing a thin piece of such a crystal through mechanical means. In some of these materials, the chemical bond anistropy is so strong that it is possible to prepare large flakes that are atomically thin . These two dimensional crystals have properties quite different from their bulk counterparts. They do have a set of discrete translation symmetries, which makes them crystals, but they only have these symmetries along two axes- there is no sense in which a one-atom-thick crystal has any out-of-plane translation symmetries. For this reason they have band structures that differ markedly from their three dimensional counterparts. As previously discussed, this process cannot be executed on every material. It depends critically on scotch tape bonding more strongly to a layer of the crystal than that layer bonds to other layers within the crystal.

Although we acknowledge this system as complex, it makes some sense to try and simplify it a little to perhaps gain some deeper insight into its operation. A glance at figure 12 suggests that there are two generalized groups of ant predators on the berry borer: the big ants and the small ants . So we can think of it as a two-predator, one-prey system. But there is an obvious indirect effect because the bigger ants negatively affect the ability of the smaller ants to be predators, as was discussed above. Depending on nest densities, it would seem that because the smaller ants prey on all three life stages of the borer and throughout the year for those that forage in infected berries on the ground, they might, in the end, be more efficient predators than the larger ants. Furthermore, the larger ants have an indirect trait-mediated effect on the smaller ants, reducing their effectiveness. One might argue that it is the existence of the smaller ants that potentially regulates the borers over the long run. Unpublished evidence even supports the idea that the berry borer actually seeks areas that are under protection from the ants, supporting the speculation of Gonthier and colleagues that the berry borer gains protection from smaller predators by preferring to attack berries under the protection of larger ants. This basic speculation was put to an unintended test in 2012. Because of a major outbreak of the coffee rust disease , the coffee landscape where we work was heavily sprayed with a combination of calcium carbonate and copper sulfate, hydroponic bucket a permitted activity for organic agriculture. In figure 13, we show the distribution of P. synanthropica as a heat map based on how many tuna fish baits placed in coffee bushes had swarms of P. synanthropica after about 30 minutes.

It is clear that in a single year, a population of thousands of nests of P. synanthropica simply disappeared. Surveys in subsequent years indicated that the smaller ants in the system began to recuperate from the reduced state they had been in, apparently because of the indirect effects of P. synanthropica . Those small ants that attack the borer within the seed, both on the tree and on the ground, especially increased over the next few years. In figure 14, we show the distribution of several of the species in a 50 × 50 meter subplot within the 45-hectare plot. Note how, during the years 2009–2012, the distribution of P. synanthropica remained relatively constant, perhaps slowly increasing in its area of dominance, at the expense of P. protensa on the ground and S. picea arboreally. Then, after the collapse of P. synanthropica between 2012 and 2013, both of those smaller species began to move into the area previously dominated by P. synanthropica. If the above speculations about how the ant community affects the borer are true, we might expect that the elimination of one of the borer’s predators would result in better overall control of the berry borer. In surveys of the berry borer in 2005 and then repeated in approximately the same area in 2018, the attack rate of the borer went from an average of about 15% of berries infected with borers to less than 1%. Insect populations are notoriously variable and respond to many cues in the environment by increasing and decreasing population numbers, frequently in unpredictable ways. Therefore, although this dramatic decline in borer numbers cannot be directly linked to the change in the ant community structure, it is nevertheless worth noting that the underlying narrative of how that community functions as a system of biological control concords perfectly with the changes observed.In the early 1980s, a specter haunted the coffee growing regions of Central America.

The infamous coffee rust disease had arrived in Brazil, and its eventual spread all the way to Mexico was expected, causing extreme worry among farmers and technical advisors. This worry was certainly justified on the basis of the history of the coffee rust disease in Asia . Great Britain’s expansion in what was then called Ceylon was qualitatively distinct from many of its previous imperial adventures. Planting what was effectively a monoculture of coffee, along with a great deal of infrastructure for the time, it was a remarkable centrally planned agricultural development plan. However, the plan effectively created ideal conditions for any disease that could get a foothold, with its virtually shadeless monoculture and networks of roads and railroads that could help distribute the fungal spores widely. When the disease arrived, it took hold and spread throughout the entire island, eventually causing a complete loss of coffee production . However, the rust scare of the 1980s Mesoamerica turned out to be a bit of a false alarm, at least until 2012. Before that year, the rust was always an irksome constraint on production, but the complete devastation that had been feared when it was discovered in the early 1980s never came to pass; it was a problem, to be sure, but not one to get overly agitated about. But then, without much warning, there was an explosion of coffee rust in the 2012–2013 cycle. Countries in the zone declared emergencies as one of their main sources of income seemed to be threatened with severe disruption. Local governments throughout the affected area provided emergency support to coffee producers and both the United Kingdom and the United States came up with significant international aid, specifically for what rapidly came to be called the most devastating emergency in the history of coffee production throughout the region . There are two ecological questions associated with this episode. First, why did the disease not become rampant for approximately 30 years after its introduction, and, second, what caused the very sudden explosion?

Although the answers to either of these questions remain enigmatic, using tools from complexity science provides us with some ideas. The relevant biology of the rust disease is well known. A windblown spore adheres to the under surface of a leaf and encounters a small amount of moisture, causing germination directly into a stoma. The mycelia grow intercellularly and produce haustoria, which penetrate into the plant cell and absorb nutrients, effectively killing the cell. As the fungus grows within the leaf tissue it eventually forms uridia that contain new spores, exiting the leaf from other stomata, causing the characteristic yellow spots on the under surface of the leaves . The transmission dynamics of the disease are dual , stackable planters with some close plant-to-plant dispersion of spores , especially when plants are close enough to touch one another, along with propagule rain from the general spore load that exists in the atmosphere, especially in areas of high concentration of coffee production, when that coffee is attacked by the rust. From the perspective of an individual coffee plant, there are two sources of rust spores: its local neighbors and the general accumulation of spores in its region—that is, from the overall spore load in the atmosphere. But it is also the case that this coffee plant and all others over a very large region contribute to the spores in the overall spore rain from the atmosphere. Given this narrative, it is easy to imagine a situation in which a generally traditional shade coffee landscape would receive a particular rate of spore rain each year and would contribute a bit to the general pool, but because the shade trees act as windbreaks, much of the spore load is never delivered to the coffee plants. One could imagine an equilibrium in which the rust disease would be endemic but not severe, partly because the wind-borne spores have limited access to the coffee trees, meaning that the increment of spore load in the general atmosphere would be limited. Focusing on the large landscape level, if the abundance of spores in the atmosphere is low, it is likely that the incidence of the disease will also be low. But each epidemic will increase the spore density in the atmosphere. The probability that a given farm will become epidemic is a function of both the spore density in the atmosphere and the dispersion rate from the atmosphere to the farm. Changing focus to the local level, the rate of spread of spores from coffee bush to coffee bush on an average individual farm will partially determine whether the rust within that farm will become epidemic. From the point of view of an individual coffee bush, the danger of being infected by a spore comes from two sources: the atmosphere and neighboring infected plants—a regional source and a local source. Imagine that a forested ecosystem is gradually deforested of both shade trees in the coffee farms and the trees in the natural forest around them, and ask what proportion of the farms could be susceptible to an epidemic of coffee rust? According to a simple model that incorporates both regional and local dispersal , the initial deforestation will generate an increase in the number of farms experiencing an epidemic. That increase is likely to be slow and steady at first, but there will be a specific point at which a critical transition will occur and a large number of farms will suddenly become highly infected. This will happen in the complete absence of any other environmental driver, such as climate change or a new more virulent strain of the disease. Indeed, one study in Costa Rica showed that the incidence of rust disease was correlated with the amount of sun coffee and pasture in the surrounding landscape. It could very well be that the sudden outbreak of coffee rust in 2012 is an example of the inevitability of surprise arising from the formality of a critical transition that we have come to associate with highly nonlinear complex systems .

A cartoon version of this theoretical process is presented in figure 16. An important component of the rust disease system, not yet completely understood, is the existence of several natural enemies of the rust . Providing an example of the sorts of ecological complexity of popular literature, the fungal disease of the first pest we discussed, the green coffee scale , is caused by the same species of fungus that, when given the chance, attacks the coffee rust fungus. That same white halo fungus that attacks the green coffee scale, now acts as a mycoparasite . Because this natural enemy is also a natural enemy of the green coffee scale, the connection to the Azteca ant became obvious early on ; Azteca creates conditions under which the scale insect becomes highly concentrated locally, which attracts the infestation of the white halo fungus and creates local hot spots of spores that disperse locally and attack the rust. Correlative evidence for this hypothesis, prior to the 2012 epidemic of rust, comes from multiple sources . Indeed, there has been considerable discussion at international conferences on the potential of L. lecanii as a spray for the rust disease. Our work suggests that partial control of the rust may naturally occur through this and other agents , although the epidemic throughout Mesoamerica in the 2012–2013 growing season shows the potential for the disease to escape such control, if, in fact, it did exist before that. It is quite a remarkable qualitative impression one gets when examining the rust and its control comparatively. It is endemic but rarely epidemic in Puerto Rico but has maintained a relatively severe status in much of Mesoamerica since 2012 . Examining coffee leaves in Mexico easily reveals the presence of L. lecanii but only after considerable searching effort, whereas in Puerto Rico, it is almost inevitable that, if one encounters the rust on a leaf, it is almost certain that one encounters L. lecanii also. What seems epidemic in Puerto Rico is the L. lecanii that seems to keep the rust under control. In addition to the white halo fungus, the larval form of a small fly, Mycodiplosis hamaelae, preys on the spores directly on the coffee leaf but probably also acts as a dispersal agent, at least locally . The coffee leaf rust continues to plague Latin American coffee farmers.