Month: September 2024

With less than 300 years of breeding, pedigrees for thousands of F. ananassa individuals have been recorded, albeit in disparate sources. To delve more deeply into the domestication history of cultivated strawberry, we assembled pedigree records from hundreds of sources and reconstructed the genealogy as deeply as possible. One of our initial motives for reconstructing the genealogy of cultivated strawberry was to identify historically important and genetically prominent ancestors of domesticated populations, in large part to guide the selection of individuals for whole-genome shotgun sequencing and DNA variant discovery, inform the development of single-nucleotide polymorphism genotyping platforms populated with octoploid genome-anchored subgenome-specific assays, and identify individuals for inclusion in genome-wide studies of biodiversity and population structure . The genetic relationships and genetic contributions of ancestors uncovered in the genealogy study described here guided the selection of individuals for downstream genomic studies that shed light on genetic variation and the genetic structure of domesticated populations worldwide . Our other early motive for reconstructing the genealogy of strawberry was to support the curation and stewardship of a historically and commercially important germplasm collection preserved at the University of California, Davis , plastic grow bag with accessions tracing to the early origins of the strawberry breeding program at the University of California, Berkeley , in the 1920s .

We sought to develop a complete picture of genetic relationships among living and extinct individuals in the California and worldwide populations, in part to assess how extinct individuals relate to living individuals preserved in public germplasm collections. Because 80% or more of the individuals we documented in the genealogy appear to be extinct, they could only be connected to living individuals through their pedigrees. One of the ways we explored ancestral interconnections between extinct and living individuals was through multivariate analyses of a combined pedigree–genomic relationship matrix estimated from genotyped and ungenotyped individuals . The holdings and history of the UCD Strawberry Germplasm Collection were shrouded in mystery when our study was initiated in 2015. The only individuals in the collection with pedigree records were publicly released and patented cultivars. The immediate challenge we faced in reconstructing the genealogy was the absence of pedigree records for 96% of the 1,287 accessions preserved in the collection, which is hereafter identified as the “California” population. To solve this problem, authenticate pedigrees, and fully reconstruct the genealogy of the California population, we applied an exclusion analysis in combination with high-density SNP genotyping . Here, we demonstrate the exceptional accuracy of diploid paternity analysis methods when applied to individuals in an allooctoploid organism genotyped with subgenome-specific SNPs on high-density arrays . Several thousand SNP markers common to the three arrays were integrated to develop a SNP profile database for the parentage analyses described here. SNPs on the 50-K and 850-K arrays are uniformly distributed across the octoploid genome and informative in octoploid populations worldwide . The 50-K SNP array harbors 1 SNP/16,200 bp, whereas the 850-K array harbors 1 SNP/953 bp, telomere-to-telomere across the 0.81-Gb octoploid genome.

The genealogies of domesticated plants, especially those with long-lived individuals, overlapping generations, and extensive migration and admixture, can be challenging to visualize and comprehend . We used Helium to visualize smaller targeted pedigrees; however, the strawberry pedigree networks we constructed and investigated were too large and mathematically complex to be effectively visualized and analyzed with Helium and other traditional hierarchical pedigree visualization approaches. Hierarchical methods often produce comprehensible insights and graphs when applied to pedigrees of individuals or small groups but yield exceedingly complex,labyrinthine graphs that are difficult to interpret when the genealogy contains a large number of individuals and lineages. We turned to social network analysis  to explore alternative approaches to search for patterns and extract information from the complex genealogy of strawberry. The pedigree networks of plants and animals share many of the features of social networks with nodes connected to one another through edges relationships. We used SNA methods, in combination with classic population genetic methods, to analyze the genealogy and develop deeper insights into the domestication history of strawberry . SNA approaches have been applied in diverse fields of study but have apparently not been applied to the problem of analyzing and characterizing pedigree networks . With SNA, narrative data are translated into relational data and summary statistics and visualized as sociograms . Here, we report insights gained from genealogical studies of domesticated strawberry populations worldwide. Our studies shed light on the complex wild ancestry of F. ananassa, the diversity of founders of domesticated populations of cultivated strawberry that have emerged over the past 300 years, and genetic relationships among extinct and extant ancestors in demographically unique domesticated populations tracing to the earliest ancestors and inter specific hybrids .

The genealogy does not account for lineages underlying what must have been millions of hybrid progeny screened in breeding programs worldwide; e.g., Johnson alone reported screening 600,000 progeny over 34 years at Driscoll’s . Cultivars are, nevertheless, an accurate barometer of global breeding activity and the only outward facing barometer of progress in strawberry breeding. When translated across the past 200 years of breeding, our selection cycle length estimates imply that the 2,656 cultivars in the genealogy of cultivated strawberry have emerged from the mathematical equivalent of only 12.9 cycles of selection . Even though offspring from 250 years of crosses have undoubtedly been screened worldwide since 1770, 15.5 years has elapsed on average between parents and offspring throughout the history of strawberry breeding . Because genetic gains are affected by selection cycle lengths, and faster generation times normally translate into greater genetic gains and an increase in the number of recombination events per unit of time , our analyses suggest that genetic gains can be broadly increased in strawberry by shortening selection cycle lengths. Genome-informed breeding and speed breeding are both geared towards that goal and have the potential to shorten selection cycle lengths and increase genetic gains . We reconstructed the genealogy of strawberry to inform the curation of a historically important germplasm collection, forensically identify the parents of individuals without pedigree records, authenticate the parents of individuals with pedigree records, shed light on the domestication history of strawberry, and retrospectively examine where we have been and how we got there. The reconstruction was greatly facilitated by the availability of outstanding SNP genotyping platforms , the development of an extensive DNA profile database to complement the pedigree database , and the application of robust and highly accurate diploid exclusion analysis methods for parent identification and pedigree authentication. We provided an open-source R code to support future parentage analyses in agricultural species. Our backward-facing genealogy study, in retrospect, pe grow bag yielded unexpected insights about the complex hybrid ancestry and breeding history of cultivated strawberry that should inspire future generations and guide where we should go from here. Our critical examination of historical selection cycle lengths was meant to be provocative and perhaps inspire the implementation of strategies for increasing breeding speed and accelerating the improvement of strawberry. We suspect that improvements can be achieved, at least in part, through changes in breeding schemes and the application of pedigree-informed predictive breeding methods. The open-source pedigree database we compiled should find broad utility in predictive breeding schemes and can be easily expanded and modified for specific breeding problems, other populations, and future analyses. Because of the depth and completeness of the pedigree records commonly available in strawberry, pedigree best linear unbiased prediction has the potential to increase genetic gains and enhance selection decisions, especially when combined with genomic prediction . The pedigree database we assembled will facilitate the application of pedigree-BLUP and identity-by-descent prediction of alleles and haplotypes , in addition to providing a solid foundation for expanding the genealogy over time.We are grateful to Clint Pumphrey, the manuscript curator of the special collections and archives of the Merrill-Cazier Library at Utah State University . Clint assisted the first author with acquiring and researching the laboratory notebooks and other records of Royce S. Bringhurst , a former faculty member and strawberry breeder at the UCD . The documents and photos associated with the collection yielded extensive pedigree records that were crucial for reconstructing the genealogy of the UCD Strawberry Breeding Program. We are equally grateful to Phillip Stewart, a strawberry breeder at Driscoll’s , for sharing copies of the UCB, pedigree records of Harold E.

Thomas , a former faculty member and strawberry breeder at UCB from 1927 to 1945. Those pedigree records greatly increased the completeness and depth of the database for the early years of the University of California Strawberry Breeding Program. The authors thank Thomas Sjulin, a former strawberry breeder at Driscoll’s , for sharing the public pedigree records he assembled over his career. Those nucleated the pedigree database we developed and were a catalyst for our study. SJK and GSC thank Robert Kerner for the computer forensic analyses that recovered several hundred pedigree records for UCD individuals from an obsolete electronic database, thus preventing the loss of those records for perpetuity. They were critical for integrating the UCD genealogy with the global genealogy for cultivated strawberry. SJK especially thanks Rachel Krevans, Matthew Chivvis, Jake Ewert, and Wesley Overson for their integrity, friendship, and steadfast support.Table grapes have become an important fresh commodity in Brazil for both internal market and exportation. Over the period of 2000–2016, Brazil presented an increase of ∼150% in table grape production, reaching around 970,000 MT in 2016 . The northern region of Paraná state is one of the main areas of table grape production. The mild winter and subtropical conditions of this region permit two crops of grapes per year, which allow Brazilian growers to time their production to coincide with market windows of other countries and compete for more advantageous prices. However, in these subtropical regions, berry ripening and harvest often occur during the rainy season, which is not ideal for Vitis vinifera cultivars because excess rain and moisture compromise the overall quality of the berries . Therefore, Brazilian table grape production is starting to incorporate American and/or hybrid grape cultivars that are better adapted to warm and rainy climates. Another disadvantage of growing table grapes in subtropical areas is that high temperatures during ripening can inhibit anthocyanin biosynthesis in the berries from V. labrusca and hybrid cultivars . This results in inadequate fruit color, and thereby a decrease in market acceptance and the potential economic value of the commodity . The seedless table grape Selection 21, a new hybrid of V. vinifera × V. labrusca recently developed by the Grape Genetic Breeding Program of Embrapa Grape and Wine, Brazil, obtained from the cross of [Arkansas 1976 × ] × “BRS Linda,” is a clear example of a cultivar that lacks red color development when grown in subtropical regions. The plant growth regulator ethephon, an ethylene-releasing agent, has long been known to improve berry color when applied at véraison . More recently, the application of –cis-abscisic acid has also been shown to stimulate anthocyanin accumulation and thereby improve berry color . S-ABA appears to be more effective than ethephon in enhancing grape color and it has other potential benefits compared to ethephon, including a shorter postharvest interval, and an exemption from tolerance in most countries. The introduction of S-ABA as an active ingredient in a commercial plant growth regulator prompted many studies on V. vinifera cultivars under temperate climate conditions. Such studies have shown that the efficacy of S-ABA varies with the cultivar , the S-ABA concentration , the time of application and the environmental conditions . Studies have shown that exogenous application of S-ABA can significantly increase the activity of a wide range of genes involved in anthocyanin biosynthesis . Most of these studies tested the effects of a single application of S-ABA before or during véraison. However, studies of the effects of S-ABA several applications at different concentrations and timings following véraison are still needed to optimize the use of this plant growth regulator in table grape cultivation . In grapes, the anthocyanin biosynthesis pathway involves multiple steps that are controlled by MYB transcription factors, such as VvMYBA1 and VvMYBA2 . In red varieties, the VvMYBA1 gene is only expressed after véraison. Both VvMYBA1 and VvMYBA2 regulate anthocyanin biosynthesis during ripening by strictly controlling the expression of the canonical UDP-glucose:flavonoid 3-Oglucosyltransferase gene .

Agricultural intensification simplifies ecosystems through management practices such as increases in agrochemical use, decreases in habitat complexity, and decreases in crop and vegetation diversity. Agricultural intensification alters functional biodiversity;in particular, reductions in habitat complexity impact the arthropod community composition, decrease arthropod diversity and reduce pest control services. Notably, biological pest control is likely the ecosystem service most affected by biodiversity loss at the local scale. In coffee agroecosystems, management intensification alters habitat complexity by impacting vegetation connectivity and structure. The management intensification gradient ranges in coffee systems from the least intensive traditional shaded “rustic system”, in which coffee grows under a diverse closed canopy of native forest, to the most intensive “sun monoculture”, which refers to rows of open unshaded coffee monoculture, that require high inputs of agrochemicals. On the shaded end of the intensification gradient, shade coffee habitats are naturally vegetatively complex, with diverse and dense shade canopies and vines and weeds that form connections between the shade trees and the coffee plants. This vegetation connectivity is an important aspect of habitat complexity that impacts species interactions at the local scale. However, while progressing along the management intensification gradient, reductions in habitat complexity, driven by decreases in shade trees, increases in herbicide use, and the clearing of vegetation between coffee plants, reduce vegetation connectivity and alter species interactions within ecological communities and the ecosystem services that they provide. Connectivity is one physical component of habitats that has a profound impact on arboreal insects and ant community structure. In the absence of connectivity, plastic square flower bucket trees are insular habitats with crown isolation that inhibits the movement of some taxa.

Connectivity in the form of lianas and nylon ropes shape the local community structure of arboreal ants, with higher ant species richness often occurring in trees that are connected artificially or vegetatively as compared with trees without these physical connections, and higher ant species coexistence occurring in trees with higher levels of naturally occurring canopy connectivity. These results also reflect the nature of ants as highly efficient foragers, known to use branches and lianas as “opportunist walkways” that provide the quickest foraging routes by allowing for faster traveling speeds through avoiding obstacles on the ground, even if these routes are not necessarily the shortest distance. The variation in texture of natural walkways, characterized as “surface roughness”, further impacts both arboreal and ground ant running speeds and foraging efficiency. Physical connections between trees are thus important structures that facilitate not only arboreal ant mobility but also their foraging success, resource recruitment efficiency, and ant-provided ecosystem services, including pest removal. Ants play an important role in the control of the coffee berry borer , the most damaging insect pest of coffee. In particular, the aggressive arboreal ant Azteca sericeasur nests in shade trees, forages on coffee shrubs, and is a keystone predator that controls the CBB. Like many arboreal ants, A. sericeasur prefers walking on branches and vegetation to avoid traveling on the ground. Given the role of A. sericeasur as a biological control agent, understanding how connectivity at the local scale impacts these ants has potential implications for coffee agroecosystem management. In Chiapas, Mexico, Jiménez-Soto et al. found that artificially increasing connectivity between A. sericeasur nests and coffee plants by tying jute string between ant nest trees and coffee plants increased the capacity for A. sericeasur to remove the CBB by throwing them off the coffee plants.

These results suggest that naturally occurring vegetation connectivity might have a similar effect as that of artificial string connectivity on A. sericeasur activity and their associated pest removal services. Our study tests and expands on this hypothesis by examining the impact of both artificial connectivity and naturally occurring vegetation connectivity on A. sericeasur activity, its ability to recruit to resources, and its removal of the CBB with a manipulative experiment. Specifically, we tested the following hypotheses: We predicted that the coffee plants with vegetation or artificial connections to the ant nest tree have higher A. sericeasur activity than that of the isolated control plants; A. sericeasur ants recruit to resources more efficiently on coffee plants with vegetation or artificial connections to the nest tree; coffee plants with vegetation or artificial connections to the nest tree have greater CBB removal rates by A. sericeasur ants; and A. sericeasur activity, resource recruitment rates, and CBB removal rates decrease with increased distance from A. sericeasur nests. This study was conducted in the Soconusco region of Chiapas, Mexico at Finca Irlanda, a shaded, 300-hectare commercial polyculture coffee plantation. The plantation is located in the Sierra Madre de Chiapas Mountains at an elevation of 1100 m.a.s.l. The average canopy cover throughout the farm is 75 percent and the majority of the plantation shade trees are of the genus Inga. The climate is semi-tropical, with the rainy season occurring between May and October. Vegetation management at Finca Irlanda frequently includes “chaporreo”, in which farmworkers periodically use machetes to clear the weeds and epiphytes that grow between coffee plants. This management practice facilitates farmworker movement between coffee plants and reduces competition between weeds and coffee plants, but in the process inadvertently eliminates vegetation connections between the coffee plants and A. sericeasur nest trees.We collected data between June and August in the summer of 2022.

Within the 300 hectares of Finca Irlanda, we selected 17 trees with active A. sericeasur nests as study sites. Each site was located at least 10 m away from any other active A. sericeasur nests to prevent overlapping ant activity, following the methodology used by Jimenez-Soto et al.. We chose six coffee plants within a 5 m radius of each A. sericeasur nesting tree for a total of 102 coffee study plants. At each nesting tree site, we selected two coffee plants for the natural vegetation connectivity treatment, two for the artificial connectivity treatment, and two as isolated control plants . For the vegetation connectivity treatment, we selected two coffee plants with existing vegetation connections. The vegetation connections were either coffee branches directly touching the A. sericeasur nest tree or coffee branches touching a secondary plant, such as a vine or epiphyte that was touching the nest tree. We selected two coffee plants for the artificial connectivity treatment, in which we tied jute strings between the point of the nest tree trunk with the most active ant foraging trail and the central trunk of each coffee plant. We ensured that there were no existing vegetation connections on these plants and that the string was the only point of connection between each coffee plant and the nest tree. For the control treatment, we selected two isolated coffee plants with no connections between the coffee plants and the nest tree. We measured the distance between the central trunk of each study coffee plant and the ant nest tree. At each site, we quantified the ant activity on the coffee plants by counting the number of A. sericeasur that passed a central point on the central trunk of each coffee plant during 1 min . The observations took place between 7:30 AM and 2 PM before the afternoon rainy period. The observations were stopped if it began to rain, as rain significantly reduces ant activity. After setting up the strings, we returned to each site between 7 and 13 days after the initial setup and re-measured ant activity on the coffee plants. To assess the impact of artificial and vegetation connectivity on prey removal by A. sericeasur, plastic plant pot we placed five dead adult female CBB on white index cards on the centraltrunk of each coffee plant . We monitored A. sericeasur interactions on the cards for one hour, ensuring that only A. sericeasur were responsible for removing CBB, and counted the number of CBB removed. Because it has already been well-documented that A. sericeasur remove live CBB from coffee plants, we used dead prey to avoid the possibility of live CBB escaping during a longer observation period. The CBB were collected from infested coffee berries in the field, then frozen for up to 5 days before use. Recruitment is understood to be an integral component of trail-following in which ant workers follow chemical foraging trails to a food source, then re-apply chemical trails until that food source is exhausted. Tuna baiting is an effective and widely used method of assessing ant recruitment in coffee agroecosystems. To assess the impact of connectivity on ant resource recruitment efficiency, we placed 1 g of canned tuna on the central trunk of each coffee plant 1 m above ground and recorded the number of A. sericeasur that recruited to each tuna bait after 20 min. To test for statistical differences in ant activity, resource recruitment efficiency, and CBB removal between the control, string, and vegetation treatment coffee plants over the 5-week experiment, we fit our data with generalized linear mixed models using the lme4 package in R. For each response variable , we included the time , the treatment method , the distance between the coffee plant and the ant nest tree , the interaction between the treatment and the time, and the interaction between the time and the distance as fixed effects.

As random effects, we modeled the coffee plant identity nested within the site to control for site variation and spatial non-independence. To assess count data , we originally fit each model to a Poisson distribution. However, to correct for observed over-dispersion, we instead modified each model to a negative binomial distribution. We observed A. sericeasur using the artificial string connections at 12 of the 17 sites and on 20 of the 33 strings placed . A. sericeasur were the only ants observed using the strings. Out of 33 vegetation treatment plants , 20 plants included primary connections , and 13 plants were connected by secondary connections . We observed A. sericeasur utilizing vegetation connections on every vegetation treatment plant. Ant activity was higher on the vegetation treatment coffee plants than on both the control treatment and the string treatment . There was a significant effect of time on ant activity for the string treatment, indicating an increase in ant activity on the string plants after connecting the strings . There was no effect of time on ant activity for either the control or vegetation treatments, indicating that there was no significant change in ant activity for those treatments over the 5-week experiment . Treatment, time, and distance all impacted the ant recruitment to tuna baits. More ants recruited to the tuna baits on the vegetation treatment plants than on the strings, and more ants recruited to the bait on string plants as compared to the control plants . The overall number of ants that recruited to the baits decreased with time post string placement on both the control and vegetation plants, but there was no significant change in the number of ants recruiting to the baits on the string treatment . The number of ants recruiting to the baits on the control and string plants declined with distance from the nest tree, but was consistent over all distances for vegetation treatment plants .Treatment, time, and distance all impacted the number of CBB removed by ants. Ants removed more CBB from the natural vegetation treatment plants than from the string plants and removed more CBB on the string plants compared to the control plants . The overall number of CBB removed from the vegetation plants decreased with time post-string placement . The number of CBB removed on the control and string plants declined with distance from the nest tree, but was consistent over all distances for vegetation treatment plants .This study asked how connectivity, occurring naturally as vegetation or artificially as string connections, influences A. sericeasur activity, foraging efficiency, and pest removal services in coffee systems. Our research demonstrates that naturally occurring vegetation connectivity and, to a lesser extent, artificial connectivity between A. sericeasur nest trees and coffee plants increased both A. sericeasur mobility and CBB removal on coffee plants. Between the control, string, and vegetation connectivity treatments, all response variables were highest on coffee plants with naturally occurring vegetation bridges between the coffee plants and the ant nest tree.

The first three inoculated time points, 6, 12, and 24 hpi, had an average percentage of mapped reads between 85 and 88%. The last two time points, 36 and 48 hpi, where microscopic and macroscopic cell death was observed only had 52 and 64% of their reads mapped respectively. The complete results for total reads, percentage of reads mapped, and percentage of uniquely mapped reads are listed in Table 4.2.The total number of annotated proteins as well as DEGs are listed in Table 4.3. There were the highest number of both up-regulated and down-regulated DEGs at 24 hpi at 14,195 and 13,237 respectively. Using a cluster analysis of differentially expressed genes , the function of unknown genes can be recognized. In the hierarchical clustering, different areas with different colors, represent different groups of the cluster of genes up and down-regulated. This analysis shows two major clusters by treatment, one composed of all the mock uninoculated samples and inoculated samples corresponding to early time points . There were a large number of upregulated enriched genes associated with the ribosome and protein processing in the endoplasmic reticulum at 48-hpi, 321 and 189 respectively . There were also 72 enriched genes associated with the ribosome down-regulated at 12-hpi . Downregulated genes associated with the spliceosome were enriched at 12 and 36-hpi . There were many common differentially expressed plant defense genes that were upregulated in P. cinnamomi infected N. benthamiana leaves when compared to rootinoculated avocado, chestnut, and eucalyptus . Specifically, PAL, Thaumatin, Allene oxide synthase, F-box proteins, and cytochrome P450 were also significantly upregulated in avocado and L. longifolia roots. Genes encoding several members of the WRKY transcription factors were up-regulated in avocado, eucalyptus, chestnut, and L. longifolia roots. Glutathione S-transferase gene was up-regulated significantly in avocado and L. longifolia roots.

These and other defense related genes commonly expressed in avocado, round flower buckets other model systems, and the N. benthamiana pathosystem support the use of N. benthamiana to investigate defense gene response to P. cinnamomi.In this study we have elucidated the gene expression in response to P. cinnamomi infection in the N. benthamiana model system. By analyzing the transcriptome of N. benthamiana with RNAseq at five critical time points during the infection it was possible to identify important defense pathways using our model system. As early as 6-hpi there is a response by the infected host. We see a significant number of genes involved in the biosynthesis of secondary metabolites up-regulated. Specifically, known defense-related biosynthesis pathways such as flavonoid, terpenoid, and the phenilpropanoid pathways were enriched. Numerous plant-pathogen interaction genes were also enriched indicating the initiation of an active defense response to the pathogen. The highest number of enriched genes were involved in the biosynthesis of secondary metabolites at both 6 and 12-hpi. There was also a significant down-regulation of genes associated with the ribosome and plant hormone signal transduction especially auxin related genes. There is also a down-regulation in photosynthesis and metabolic pathways indicative of a decrease in resources allocated to growth and energy production in response to pathogen detection. The DEGs analysis confirms this early defense response with up-regulated hormone signaling, transcription factors, pathogen related genes, and resistance genes. At 24 hpi the KEGG enriched terms show a decrease in carbon fixation and metabolism which supports the allocation of resources towards plant defense. Salicylic acid binding is also down-regulated 5-fold which could be the result of pathogen effectors subverting the defense response. At the same time, we found transcription factors, PR genes, R genes, and anti-fungal genes that were up-regulated over 10-fold. Although P. cinnamomi is attempting to subvert the host defenses, the extensive colonization and intracellular growth at this stage of the infection has induced an extreme response in N. benthamiana.

At 36 hpi we found that besides significant up regulation in the plantpathogen interaction KEGG enrichment pathway there was up regulation of genes involved in endocytosis and phagosomes. Interestingly this is the time point where plant cell death becomes apparent in previously stained images of the infection process and is thought to be the stage where P. cinnamomi becomes necrotrophic in its infection strategy. There is also a 10-fold down regulation of SA binding and 5 to 10- fold up regulation of the JA pathway that further supports the necrotrophic infection occurring at this stage. At 48 hpi the KEGG pathway shows that 321 unigenes are involved in the up regulation of ribosome function. At the same time 770 unigenes are involved in the down regulation of metabolic pathways. Numerous defense genes are also being highly expressed at this time point including the continued up-regulation of the JA pathway. PAMP-triggered immunity is the plants first layer of defense against plant pathogens. Plants have developed pattern recognition receptors that initiate a defense response before the pathogen is able to infect the plant. This early PTI response is at a time in our model system when many of the encysted zoospores on the inoculated leaf surface haven’t germinated and there is no intracellular penetration . PTI response is linked to reactive oxygen species production, lignin and callose reinforcement, and the up-regulation of pathogenesis-related genes. Genes encoding Glutathione S-transferase, which serves to protect plant cells from ROS production was significantly up-regulated in our system as well as previously identified as being up-regulated during infection in Arabidopsis, Z. mays, and avocado . PR-genes up-regulated significantly in N. benthamiana in response to P. cinnamomi infection included; PR-1, PR-4 , PR-5 , and PR-9 . PR-5 was also up-regulated in infected avocado roots and PR-1 and PR-5 were up-regulated in eucalyptus roots . Hormone signaling plays an important role in a plants response to various pathogens and the pathway initiated varies depending on the type of pathogen. The jasmonic acid and ethylene signaling pathways are normally initiated in response to necrotrophic pathogens.

The cytochrome P450 super family is the largest enzymatic protein family in plants . CYP genes are involved in hormone signaling and associated with the JA pathway . CYP has been described as a JA-responsive gene which was up-regulated 37.75-fold in L. longifolia . Numerous genes in this family were up-regulated in all of the five time points we analyzed. Up-regulation of CYP has also been found in inoculated avocado . Some interesting and less well described anti-fungal genes in the CYP family, premnaspirodiene oxygenase and aristolochene synthase were identified that would be good candidates for further analysis. F-box proteins associated with the JA pathway were upregulated in 4 out of 5 of the time points in our system. The up-regulation of this gene in response to P. cinnamomi infection has also been discovered in avocado as well as numerous other model systems indicating a similar response among these different plants. Allene oxide synthase genes involved in JA biosynthesis , were also up-regulated in our system. JA response is traditionally associated with necrotrophic pathogen defense, plastic flower buckets wholesale but recent studies have shown that biotrophic pathogens such as Plasmopara viticola and hemi-biotrophs like P. infestans and P. cinnamomi can also trigger the activation of a JA triggered response. There were also some differentially expressed genes that are normally associated with the SA pathway discovered in the N. benthamiana system including up-regulated PAL genes and down-regulated SA binding genes. PAL is the key enzyme for the phenylpropanoid pathway which is involved in SA biosynthesis, lignin, and antimicrobial compounds such as flavonoids and phytoalexins . Auxin signaling has been shown to play and important role in the induction of resistance to P. cinnamomi . Plants using more than one defense pathway in response to P. cinnamomi infection has been seen previously in avocado and L. longifolia but was not found in Z. mays . Interestingly, genes encoding WRKY transcription factors were significantly upregulated at all of the time points in our system. In plants, WRKY transcription factors are encoded by a large family of genes, and they are involved in abiotic and biotic stress and are activated by pathogen perception . WRKY genes have been previously shown to be direct transcriptional targets of NPR1 in response to SA abundance . The significant up-regulation of WRKY transcription factors in our model system may indicate that there is some JA/ SA crosstalk in the infected samples. Up regulation of WRKY transcription factors in response to P. cinnamomi infection was also found in eucalyptus, chestnut, and L. longifolia roots. . WRKY51 transcription factor was selected as an ideal candidate for functional experiments because of its consistent up-regulated expression from 12 to 48 hpi shown in the RNAseq data and the uniform results during the qPCR validation. For the many reasons listed above WRKY51 transcription factor was chosen to functionally validate in our N. benthamiana model system. The transient over-expression of the WRKY51 construct was carefully timed through numerous experiments to discover at what point, if any, in comparison with the P. cinnamomi infection would the expression of this transcription factor have the greatest effect on the colonization of the pathogen. Transient expression of the WRKY51 protein 3 hours before inoculation with P. cinnamomi zoospores produced the clearest phenotype difference between the experimental and control groups. This early transient expression may induce an early defense response before the pathogen has a chance to colonize the host.

The RNAseq data in this study and others, and the functional assay using WRKY51 confirm that a close analysis of its expression in avocado is warranted. The RNAseq data as well as the functional work in this study provide a wealth of information concerning host defense response to P. cinnamomi infection. It is important however, to begin to make connections back to avocado which is the economically important crop we are interested in understanding more completely. By using the data obtained in this study and developing functional experiments that overcome the limitations associated with tree crops such as avocado we will be able to better address the long-term breeding concerns of avocado growers. Genes identified in our model system such as cytochrome P450 or identified and validated such as WRKY51 canbe used for marker assisted breeding. The avocado qPCR data for cytochrome P450 is an important first step in the goal to learn more about avocado defense gene response directly. The expression of cytochrome P450 in inoculated detached avocado leaves was similar to the expression in detached N. benthamiana leaves in both PS.54 and Dusa® rootstocks. Since cytochrome P450 has already been identified to be significantly up-regulated in P. cinnamomi infected avocado roots , it is reasonable to infer that this defense gene is similarly expressed in both roots and shoots. These universally expressed plant defense genes will provide vital information for resistance breeding projects in avocado. The next step is to find a functionally validated defense gene in our model system that is differentially expressed in the susceptible and tolerant avocado varieties and also transiently over express the avocado candidate genes homologs in N. benthamiana for functional validation. This would be an ideal candidate for marker assisted breeding. Using our model system, it is possible to identify such a candidate.Plant-parasitic nematodes infect a broad range of commercially important crop families such as the Solanaceae , Fabaceae , Malvaceae , Amaranthaceae , and Poaceae , causing an estimated annual loss of $80 billion USD . The most economically important group of PPNs are sedentary endoparasites, including root-knot nematodes and cyst nematodes . Sedentary endoparasites induce the formation of permanent feeding cells that provide specialized nutrient sources for nematodes . Infective second-stage juveniles of RKNs predominantly invade near the root tip and then migrate intercellularly toward the apical meristematic region without crossing the endodermis, making a U-turn to enter the vascular cylinder where they induce several giant cells as a feeding site by stimulating the redifferentiation of root cells into multi-nucleate giant cells by repeated nuclear divisions without cytoplasmic division. After maturation, adult RKN females lay eggs in a gelatinous egg mass on or below the surface of the root . In contrast, CNs move destructively through cells into the vascular cylinder, select a single cell, and form a syncytium as a feeding site by local dissolution of cell walls and protoplast fusion of neighboring cells. A CN female produces hundreds of eggs and its body forms a cyst that can protect the eggs for many years in the soil .

The need for additional replication may also have been reduced by using elevated amounts of genomic DNA and the use of end-labeling rather than BioPrime may have increased the reliability of calls. The protocol used for hybridization of lettuce genomic DNA was also subsequently highly effective for pepper and other Solanaceae. Furthermore, the use of genomic DNA is a desirable target because SFPs identified using cDNA may be a result of alternate splicing or gene expression differences. Rostoks et al. indicated that 40% of the SFPs they identified may have been falsely called and partially explained them as being mRNA structural variants. They also reported a high predicted false positive rate of 22% for SFPs detectedusing genomic DNA. We concluded that fragmented, end-labeled genomic DNA provided a suitable target for detection of polymorphisms while reducing false positive sequence polymorphism . The overlapping tile design increases the likelihood of detecting polymorphisms due to redundancy at individual positions, coverage along the contigs and optimal position of the SNP within a probe. Furthermore, the number of probes and hence the possible genome coverage was increased by substituting mismatch probes with AG probes for background correction and normalization of data. Because the peripheral 1 to 6 bases of a 25 bp oligonucleotide are less sensitive than the central bases, in terms of detecting sequence polymorphisms, procona system the tiling strategy reduces the loss of coverage due to probe position. The number and reliability of SPP calls in our experiments demonstrates that the overlapping tiling array design has improved coverage, sensitivity and specificity to detect polymorphisms.

SPP calls were validated using several approaches. The data from the two pair-wise comparisons yielded 20 to 41 thousand and 27 to 40 thousand SPPs respectively, depending on the criteria used for specificity and sensitivity. When SPPs from MSA and SFPdev were compared to the 51,552 SNPs detected between RNAseq reads of Salinas and US96UC23, 61.5% and 57.8% were found in or within at least 8 bp of the SPP range respectively, similar to that described by Gresham et al.. However, because of the high FDR associated with duplicated sequences, SPPs that were found to have a duplicated locus within the chip assembly, the gene space assembly or the genome assembly were removed from consideration; one third of the SPPs called that had duplicated loci did not contain a SNP in any of our validation tests. These identified SPPs likely were due to differences between paralogs rather than alleles at a single locus. Due to the increased redundancy provided by the mapping population of 213 RILs compared to the pair-wise comparison of the parents, SPPs in the SFPdev and MSA pair-wise comparisons that coincided with SPP mapped by Truco et al. but were absent of a SNP were considered real. Removal of duplicated loci and inclusion of mapped SPPs provided a balance between false positive and false negative rates and allowed us to optimize FDR while still discovering a high number of SPPs. Taking into consideration the lower observed FDR we concluded that the MSA method performed best as a pairwise comparison; however using multiple detection methods would yield a higher confidence in the subset of SPPs identified by both methods. The SPPs identified in the diversity panel that were polymorphic between L. sativa cv. Salinas and L. serriola acc. US96UC23 showed a low FDR. However, as a result of the filtering, sensitivity of this analysis was reduced compared to the two-genotype analyses by MSA and SFPdev. Specific analysis of the DP data for regions containing known SNPs showed that SFPdev values would have been significant in a pair-wise comparison, between SAL and SER but due to inclusion of data from all genotypes in the DP, the two were not called as polymorphic . The lack of some called SPPs in the DP may be due to larger genetic differences between L. perennis, L. virosa, or L. saligna relative to L. serriola and L. sativa. As a result of smaller hybridization differences between the more closely related genotypes, genotypes differing at a locus may have been grouped together reducing the number of SPPs called between the two genotypes.

Consequently, the DP analysis showed a lower false positive rate, but a higher false negative rate when comparing SAL and SER to sequence and mapping data. As part of our goal was to investigate the diversity and relationships of the genotypes in the DP, SPPs identified by the DP analysis were evaluated. Removal of SPPs in duplicated regions with inconsistent data or missing data was a reasonable method of removing unreliable data as these data may be from poorly performing probes in one or all replicates, heterozygous loci, paralogous genes or deleted genes. There was not a large difference in the observed FDRs for the three SPFdev cutoff values for the DP analysis; so in order to maximize the number of markers used in our phylogenetic analysis and principal component analysis, we used the least stringent cutoff value of 1.2. As the assumptions for analysis with the PHYLIP [21] package were not violated with the large number of markers, they were left as independent. To meet the constraints of the PC analysis software, markers were limited to those that were mapped. The markers discovered in our DP analysis were used to generate a phylogenetic tree showing species separation with 100% boot strap support. L. virosa and L. saligna are sexually incompatible species with L. sativa and appear to be more closely related to each other than to other species in the DP. Our data supports the conclusion by Kesseli et al., that these two species are not progenitors of L. sativa. By limiting markers to those polymorphic within cultivated lettuce we are able to separate most genotypes into classes representing each of the plant types. The butterhead type formed a distinct clade from the iceberg and cos types with 100% bootstrap support. However, the leafy type and the Batavia type both showed a wide distribution across the L. sativa clade. This is not unexpected and may reflect admixture between types during breeding programs. Alternatively, this distribution may indicate that these types are artificial polyphyletic groups based on loose morphological criteria. The leafy types are non-heading with a broad range of leaf morphology. Batavia types vary from heading to non-heading phenotypes. Batavia and iceberg cultivars are both considered crisphead types; however our phylogenetic and PC analyses showed that the two did not cluster together and are significantly different from each other . Rapid advancements in sequencing technology today are changing the methods for genetic analyses. Microarray technology presented in this paper yielded an in depth analysis of diversity for lettuce germplasm separating even closely related lines such as the crisp head class. It also has potentially several other uses including: detection of copy number variants, splice site identification, expression analysis or use with other species within the Compositae. TheSPPs identified in this study were highly reproducible and showed similar false positive results to current sequencing methods in the literature. This technology has also been used to create an ultra-dense, inter-specific genetic map between L. sativa cv. Salinas and L. serriola acc. US96UC23 to dissect phenotypic traits as well as validate and align genomic assemblies of lettuce into chromosomal linkage groups.Managing urban runoff and its associated pollutants is one of the most challenging environmental issues facing urban landscape management. The conversion of naturally pervious land surfaces to buildings, roads, parking lots, and other impervious surfaces results in a rapid surface runoff response for both time of concentration and peak flow. Impervious land surfaces adversely impact the quantity and quality of surface runoff because of their effects on surface water retention, infiltration, and contaminant fate and transport.

Large volumes of storm runoff from urbanized areas cause flooding, sewer system overflows, water pollution, groundwater recharge deficits, habitat destruction, beach closures, toxicity to aquatic organisms, procona valencia buckets and groundwater contamination. Traditional urban runoff management focuses on removing the surface runoff from urban areas as soon as possible to protect public safety. However, as excess surface water is quickly drained from urban areas, it is no longer available for recharging groundwater, irrigating urban landscapes, sustaining wildlife habitat and other uses. Green infrastructure uses natural or engineered systems that mimic natural processes to control storm water runoff. For example, traditional detention ponds have been widely used to treat storm runoff and permeable paving promotes infiltration of rain where it falls. Importantly, decentralized green infrastructure strategies control runoff and contaminants at their source. Vegetation is a green infrastructure strategy that can play an important role in surface runoff management . Large-scale tree planting programs have been established in many cities to mitigate the urban heat island effect, improve urban air quality, and reduce and treat urban runoff . There are municipal storm water credit programs in a growing number of cities that promote retaining existing tree canopy, as well as planting new trees . Although these programs encourage planning and management of urban forests to reduce runoff impacts , fertilizer is required to promote plant growth, and these added nutrients may contribute to contamination of surface runoff. Thus, reducing nutrients in storm runoff is a challenging task for landscape and water managers. Bioswales are shallow drainage courses that are filled with vegetation, compost, and/or riprap. As a part of the surface runoff flow path, they are designed to maximize the time water spends in the swale, which aids in the trapping and breakdown of certain pollutants. Bioswales have been widely recognized as an effective decentralized stormwater BMP to control urban runoff . Their effects are threefold; vegetation intercepts rainfall reducing net precipitation; plant uptake of water via transpiration reduces soil moisture, thereby increasing subsurface water storage capacity, and root channels improve infiltration . New bioswales are being developed for harvesting surface runoff and supporting urban tree growth. Bioswales that integrate engineered soil mixes and vegetation are being used to enhance treatment and storage of surface runoff . The composition of ESMs varies widely, from simple mixtures of stones and native soil to patented commercial products . Highly porous ESM mixes provide ample infiltration and pore space for temporary storage of surface runoff. Also, they support tree growth by providing more water and aeration to roots than compacted native soil alone. ESMs can reduce conflicts between surface roots and sidewalks by promoting deeper rooting systems. In California alone, over $70 million is spent annually to remediate damage by shallow tree roots to sidewalks, curbs and gutters, and street pavement . In Davis, California, a bioswale installed next to a parking lot reduced runoff from the parking lot by 88.8% and the total pollutant loading by 95.4% during the nearly two year monitoring period . Furthermore, a bioswale installed next to a turf grass patch at the University of California-Davis campus eliminated dry weather runoff from an irrigated urban landscape. The ESM used in these studies offered several advantages over other ESMs because the main structural element was locally quarried and relatively inexpensive lava rock . This ESM had a high porosity, high infiltration rate, and a high water storage capacity . The lava rock had many interstitial pores and a high surface area to volume ratio. It effectively fostered the growth of biofilms that retain nutrients and degrade organic pollutants. Because vegetated bioswale research is in its infancy, very few studies have monitored vegetation growth and its impacts on bioswale performance. Moreover, evaluation of system performance is generally conducted before vegetation is fully established . In contrast, this study evaluated the effectiveness of two bioswales on surface runoff reduction, pollutant reduction, and tree growth eight years after construction. The control bioswale contained native soil and the treatment contained an ESM. At the time of this study, the trees in the control and treatment bioswales were fully established and approaching mature size. Measurements recorded the differences in surface runoff dynamics and pollutant reduction rates, as well as tree and shrub growth. This study provides new information on the long-term effectiveness of engineered bioswales in a region with a Mediterranean climate. The water collection system was installed in 2007 to collect composited samples from natural runoff . In this study, surface runoff samples from the control site were collected at a high frequency using grab samples to better observe pollutant concentration dynamics for each experiment throughout a storm hydrograph.

The ant has several direct and indirect natural enemies, any one of which, or any combination thereof, could form the basis for the control that must occur to prevent the ant from taking over every shade tree in the plantation. One possibility we have suggested is that the phorid flies, known to reduce ant foraging activity, act as one suppressor. The reduction of foraging activity in the presence of phorids suggests that the latter could cause an ant colony to disappear, by either dying of starvation or being so harassed that the queen moves the nest to another site . A simple cellular automata model based on the natural history of the system captures the essential features of the clustering patterns of this ant . Furthermore, it has frequently been suggested that this sort of dynamic should lead to a power function distribution of the sizes of the clusters . As expected, the distribution of cluster sizes in our plot does follow a power function, as do the cluster sizes predicted by the CA model . It thus may be the case that the spatial pattern of the Azteca ants in coffee plantations forms by the same general rules that govern the formation of the spots of the jaguar or the stripes of the tiger, as suggested by the fundamental Turing process .As noted above, the relationship between A. sericeasur and the hemipteran C. viridis is a classic mutualism . While tending the scales, the ants also protect them from natural enemies, including parasitoids and at least two coccinellid predators, Azya orbigera and Diomus sp. . It is notable that these two coccinellid beetles appear to divide the habitat spatially, blueberry in pot with Diomus sp. able to feed on scales when they are separated in space from the local scale densities surrounding A. sericeasur nests while A. orbigera concentrate on those local densities near A. sericeasur nests.

Other ants are involved in tending the scales [especially the arboreal foraging but ground nesting Pheidole synanthropica , but including perhaps a dozen other species] but only on coffee plants that are not occupied by A. sericeasur . As is generally the case, coccinellid predators of hemipterans tended by ants face a dilemma; though adults can fly some distance to locate the isolated hemipterans that are not tended by ants, larvae are less mobile and need local concentrations of hemipterans to survive. However, the only place those high concentrations occur is where they are tended by ants. Consequently, the larvae of the coccinellid beetles, faced with the aggressive behavior of the protective ants, have evolved protective mechanisms against the ants, as noted above. In the network contained in the coffee system, Diomus larvae appear to engage in chemical mimicry that renders them invisible to the ants , and A. orbigera larvae are covered with waxy filaments, creating a barrier to ant attack . This arrangement provides a spatially explicit form of biological control in that the adult beetles range widely and consume hemipterans over a large area, but the larvae require the local concentrations of hemipterans that are provided only when under protection from ant mutualists, an ecosystem service provided by a simple level of ecological complexity . This also represents a spatially explicit community organization in that the ant P. synanthropica tends the same hemipterans, but never generates an extremely high density of the latter. The consequence is that when A. sericeasur searches for an alternative nesting site to escape its enemies, the residual concentrations of C. viridis supported by P. synanthropica provide them with an initial population of this key hemipteran mutualist. The alternative predator, Diomus sp., is spatially restricted to these less dense congregations of hemipterans but also participates in their overall control. Thus a spatially explicit organization of these interactions generates a unique community structure.As discussed in the section on trophic interactions, one of the main biological control agents of H. vastatrix is the white halo fungus, L. lecanii, which is commonly found in coffee plantations, especially associated with the hemipteran C. viridis when tended by Azteca ants . Because of this basic natural history , an obvious expectation is that the coffee rust disease incidence should be negatively correlated with the presence of ant nests because it is only under the protection of ants that C. viridis reaches densities high enough to attract the epizootics of the white halo fungus.

This is precisely what was found at three spatial scales. First, at a scale of 15 m, we reported a negative correlation between rust incidence and the distance to a coffee plant in which an epizootic of the white halo had killed all the hemipterans the previous year . Second, rust incidence data from plots approximately 50 m × 50 m show a negative correlation between rust incidence and coffee plants close to Azteca nests, although the R2 value was low, suggesting that many other factors affect the incidence of the disease . Finally, rust incidence data at a large scale similarly show a weak negative correlation between rust incidence and Azteca sites . It is notable that discerning the effect of L. lecanii first was facilitated by its relationship to the ant/hemipteran mutualism and thus its expected spatial pattern owing to the association of the latter with Azteca ants. At least eight other fungal pathogens are known through laboratory assessments to attack the causal agent of the coffee rust disease , yet finding spatial correlations that would indicate effectiveness in the field is not possible because of a lack of known spatial associations with other organisms. The essential ecological features of this disease are implicated in spatial ecology due to long distance dispersal by wind, local dispersal by touch and splash, mycoparasites, and other potential antagonists, and the need for a droplet of water for germination . The essential sociopolitical features include economic and political forces that cause coffee farming to either be undertaken or abandoned in a whole region, producing yet a larger spatial component of the system .Bunch grapes , notably European , are considered among the major fruit crops worldwide, producing roughly 70–80 million tons each year . Cultivars of V. vinifera L. are used for wine, juice, and table grape production. Grape berries are classified as nonclimacteric fruits, exhibiting a double-sigmoid developmental pattern with two rapid growth phases: the berry formation and the ripening phase , separated by an intermediate lag phase called the green plateau . The exponential increase in berry size characterizes both growth stages , but not the lag one .

During phases and , also known as immature stages, organic acids, mainly tartrate and malate, accumulate leading to induction of acidity levels . At the end of the lag phase, a step-change point takes place known as veraison, where acidity starts to decline while sugars, mostly glucose and fructose, as well as anthocyanins in colored varieties, increase. Of particular interest are phenolic compounds, which are major and ubiquitous plant secondary metabolites derived from the shikimate/phenylpropanoid and polyketide pathways, with three utmost categories: proanthocyanidins , also known as condensed tannins, the gallo- and ellagitannins , and the phlorotannins . Such diversity of polyphenols, with more than 8000 structural variants, bestows them a wide range of biological functions ranging from growth, development, and protection inside the plant to, to some extent, human-related issues . In grapevines, the accumulation pattern of phenolic compounds, plastic planters wholesale along with the aforementioned berry attributes, distinguishes each of the berry phases throughout berry development . Indeed, berry quality and sensory characteristics are notably defined by its polyphenol content . Remarkably, astringency is among the hardest sensory traits to depict and interpret as many intricate processes underpinning its perception . For instance, a sensory characterisation of the astringency of 11 varietals of Italian red wine revealed that neither total phenols nor PAs can predict how all astringency subtleties will be perceived . It is worth noting that the amounts, compositions, and proportions of polyphenols in a given species may vary widely depending on several factors, such as genotypic variations, developmental stages, and environmental circumstances . Scarlet Royal is a mid-season ripening table grape variety, producing seedless, red-skinned, oval-shaped, firm, and moderate to large berries with a sweet to neutral flavor . In the San Joaquin Valley, California, it typically ripens in mid to late August, filling the harvest window between Flame Seedless and Crimson Seedless, and has thus become a very popular red table grape variety in California. However, an undesirable astringent taste has been observed occasionally in some cases. In fact, the economic value of grapevines depends substantially on the environmental conditions, including climate, soil, cultural practices, cultivar, and rootstock. Hence, the term “terroir” is used in viticulture to describe the effect of such an interactive ecosystem on grapevine and wine quality . The current study aimed to understand the underlying mechanism of astringency development in Scarlet Royal berries at two contrasting vineyards . The first location produces well-colored, non-astringent berries; however, the second site yields astringent taste, poorly colored berries . The data showed a large variation in berry astringency within the same vineyard and from year to year. The data illustrated that the divergence in berry astringency stemmed from alterations in its polyphenol composition , most notably tannins. Additionally, the ripening stage was the most distinguishing platform for such variation between both vineyards. We were able to determine the tannins’ threshold level that causes the Scarlet Royal astringency taste to be ~ 400 mg/L. Given the changes in the levels of polyphenols during berry ripening, the question was raised: what is the mechanism governing the distinctive tannins accumulation pattern between V7-berries and V9-berries, and hence astringency diversity? To answer this question, RNA-seq data generated at one ripening timepoint was associated to the changes in polyphenolic levels using a systems biology approach, WGCNA .

The module-trait association analysis positively correlated the key flavonoid/PAs biosynthetic genes with the accumulation of tannins, catechin, and quercetin glycosides exclusively in V9-berries. The modulation of the berry’s transcriptomic profile is concomitant with its polyphenols’ composition, which finally disturbs berry quality, including astringency levels.Five-year-old V. vinifera cv. Scarlet Royal grafted on Freedom rootstock was chosen for its berry astringency diversity at two commercial vineyards located in Delano, San Joaquin Valley, California, USA. Vineyards were located at a close distance of 10 km, and the local weather conditions during the two seasons were collected from the Delano CIMIS weather station . Both vineyards were planted at the spacing of 2.44 and 3.66 m in an open gable trellis supporting system with East-West row orientation. Vines were pruned in a Quadrilateral cordon training with 7–8 spurs left on each cordon during the winter pruning. In addition, general UC guidelines practices were applied in both vineyard. Starting from veraison and until the end of the season, during two consecutive years . During the first year, sampling dates were July 8th , August 1st , August 10th , September 9th , September 15th , and October 19th ; and for the second year, sampling dates were: July 15th , August 10th , August 25th , September 10th , September 29th , and October 21st . Sampling dates varied from the first to the second year due to the vineyard’s accessibility. At each sampling point, two sets of fifty berries were collected periodically. The first set was used to measure the berry weight, and then these berries were macerated in an electric blender, filtered through a paper towel, and an aliquot of juice was used to determine soluble solids , pH, and titratable acidity . Soluble solids were determined using a tabletop Milwaukee MA871-BOX digital refractometer . The TA and pH were determined by titrating a 40 mL aliquot of juice with 0.1 N NaOH to a pH of 8.2 using an automatic titrator Excellence T5 . Another random 50 berries from each replicate were collected for color, tannins, and phenolic compounds and sent immediately in a cooler to EST laboratories. At harvest, which was during the month of September, an extra set of samples was collected and promptly frozen in liquid nitrogen and stored at −80°C for subsequent analysis, including RNA extraction and gene expression studies. Harvest time was determined by the growers, and the marketable clusters were picked based on the color, and yield was determined from the three harvest dates.At bloom, fifty leaves from each replicate were collected, resulting in a total of 200 leaves from each vineyard, for nutrient analysis.

Among work crews in the Santa Maria Valley we find mestizo campesinos, Mixtec and Zapotec Indians, and Mexican urbanites from, for example, Mexico City, Guadalajara, and Monterrey. We have, to be sure, identified school teachers and university graduates laboring in the fields. An examination of the valley’s agricultural labor force from the perspective of crops, as we did above, provides vital information regarding the number and flow of workers, but it reveals little about the labor force itself. To capture meaningful information on farm workers that will enable the observer to recognize behavioral regularities, educe patterns, and formulate typologies, it is necessary to observe and query the farm worker directly. We propose to accomplish this here by focusing attention on three fundamental circumstances regarding the farm worker’s life: where does s/he keep a permanent home; what is the nature of the family that inhabits that home; and what role does s/he play in the household. Answers to these three queries elicited from farm workers observed and interviewed in the valley’s fruit and vegetable fields during the 1993 campaign, allow us to distinguish five distinct types of farm workers and farm worker families from the vast and increasingly diverse universe of farm workers that people the Santa Maria Valley: the immigrant worker who has settled permanently in the valley and severed most economic ties and responsibilities with the home community in Mexico; the binational worker who maintains two functional homes, one on each side of the border, and who constantly moves back and forth between them; the Mexico-based migrant who periodically leaves home and family in search of employment and wages; the border migrantócommuterówho, using a home base in the United States-Mexico border area, collection pot accesses an assortment of job opportunities in both countries; and the seemingly single, unattached, “homeless” migrant who spontaneously and unsolicited appears in the valley looking for work.

A review of the circumstances that govern the lives of these farm workers, aside from providing interesting insights and improved understanding, allows us to identify and highlight some of the challenges and impediments that exist to correctly detect and enumerate them by, among other interested parties, the Census Bureau. Before undertaking the description and examination of the five categories of farm workers enumerated above, it is necessary to make two clarifications regarding limitations of the proposed typology. First, although the five types may suggest the logical stages of a migrationimmigration continuum, they are most definitely not. Each, in fact, represents an outcome in itself; an arrangement arrived at by design on the part of the farm worker and not a step in a process leading to settlement. Second, the described outcomes are at best temporary, passing adjustments to an ever-changing and highly unpredictable environment, one which is not only the product of agriculture’s inherent uncertainties but which is also encumbered by recent, momentous developments. Among those developments responsible for propelling change to a state of almost perpetual, unrelenting flux, to mention only the most obvious, are the rapid transformation of California agriculture and its employment practices, the never-ending changes to immigration laws and vacillating if not contradictory enforcement measures, and the changing conditions in Mexico and in the farm workers’ home communities which can either inhibit or foster migratory practices. It would be venturesome and inappropriate, therefore, to claim that the proposed characterizations represent more than current adaptations to current conditions which may change inadvertently and, once again, force farm workers and farm employers to hastily rethink and readjust their current modi operandi. As noted above, over 10,000 immigrant farm workers have settled in the Santa Maria Valley.

Many have done it permanently, which means they have relinquished their place and position in the home community, severed economic ties and responsibilities with the home-based family, and transplanted dependents to the valley. Immigrant farm workers often travel to Mexico to visit family and friends, sometimes on a regular annual schedule, but their roots are now fixed in Santa Maria. One way of ascertaining that permanent settlement has in effect taken place is when the producer and consumer components of a given domestic group are living together in the valley on the basis of locally derived income and wages. The vast majority of Santa Maria’s immigrant families come from just three states located in the central part of Mexico: Michoacan, Jalisco, and Guanajuato. The others are from northern border states such as Durango and Chihuahua , Mexico City , and the southern state of Oaxaca . Starting in 1964, a succession of at least three immigration waves populated the valley with its current mass of settled farm workers. Although prior to 1964 some farm workers had already settled in the valley forming small, marginal colonias or barrios within the towns of Guadalupe and Santa Maria, it was the elimination of the program that actually precipitated the first important movement of ex-braceros towards settlement. This action was enthusiastically urged and even abetted by local growers who feared they would otherwise lose access to their labor supply and, especially, their most skilled, trusted, and reliable workers. A second wave in 1975-1985 accompanied the expansion of high-value, labor-intensive, specialty crops which, as already discussed, created a bounty of new farm jobs with longer employment seasons. Growers once again encouraged and helped migrant employees to settle in order to ensure the presence and availability of a stable, reliable labor supply to tend valuable and highly perishable farm commodities. The third and most recent wave was prompted by IRCA and its special provisions for farm workers which were designed specifically to accommodate the interests and needs of the agricultural industry.

IRCA accomplished two things in the Santa Maria Valley: On one hand, it created a unique opportunity for many settled yet undocumented/unauthorized immigrants from earlier waves to legalize; and, on the other, it encouraged a new cohort of migrant farm workers to emulate the experience of preceding generations by also settling down. Surveys conducted in 1991 and 1993 among fruit and vegetable workers in the valley reveal that immigrants enjoy the best farm jobs, either as skilled full-time employees or in vegetable harvest crews which offer nearly year-round intermittent jobs. In fact, 74 percent of all immigrant farm workers are employed by the vegetable industry. Typically, for example, a broccoli cutter earns $1,000 to $1,200 monthly during at least nine to ten months of the year; while, in contrast, a strawberry picker earns $500 to $800 monthly during, at best, five to six months of the year. Vegetable employment and wages, in short, allow workers to minimally provide for a family living in the valley, while strawberry employment and wages do not. Immigrant families, moreover, are typically large and contain multiple wage earners who can assemble a sizable annual income by sharing resources. A preferred arrangement is to place the household head in year-round employment while the spouse and other family members find occasional part-time jobs weeding and thinning vegetable crops and perhaps harvesting strawberries in the spring and summer. An immigrant family who cannot place one or more workers in year-round or near year-round jobs, in contrast, 10 plastic plant pots must deploy all its available workers, including children, during the short but intense strawberry harvest to amass sufficient income to carry them over into the next employment season. Valley immigrants only rarely leave the area to seek employment elsewhere during both expected and unexpected periods of high unemployment and underemployment but rely on unemployment insurance and occasional odd jobs to tie them over. Immigrant families are not only large, but nearly 45 percent of them are extended; that is, they are made up of one nuclear family and at least one arrimado -usually a live-in relative. Many extended groups include two or more nuclear families with arrimados who share income, expenses, and household responsibilities. About one-third of the settled families, particularly those who arrived with the first waves, own their homes, while one-half of the families who rent have lived at the same address for at least three years. It is, therefore, a relatively stable population. Newcomers, those who arrived with the last wave, experience a more precarious existence and, as a result, frequently change domicile. There is, for instance, an observable annual concentration-dispersion cycle which corresponds with periods of high and low employment; that is, in bad times several families will converge, actually crowd, into a shared apartment, dispersing into separate homes as soon as better times return. Immigrant homes, finally, contain a considerable number of “visitors” who are either family and friends from the home community in Mexico or paying boarders. Settled families, in fact, represent a sort of haven for seasonal migrants, especially kin, who receive shelter and assistance while they remain in the valley during their annual trek from Mexico. On the other hand, by letting rooms, converted garages and other home facilities to non-kin during the farm employment peaks, immigrant families earn additional revenue with which to supplement an always insufficient farm income.

Settled immigrant families, in contrast with all other farm workers, lead relatively stable existences in the valley. They, in fact, enjoy a greater degree of employment security and many have set up permanent residences. As such, it would appear that settled families should not pose serious difficulties or obstacles to enumeration efforts. To accept this as a sound conclusion, however, would be a grave mistake. Settled families, to begin, harbor a significant number of unauthorized/undocumented immigrants who need to be protected from detection. Although IRCA amnesty provisions allowed many long-term undocumented immigrants to legalize, it forced many others who did not qualify for any of the programs, who were unable to assemble the required documents, or who just simply did not understand the new law to remain undocumented. IRCA also enticed many regular sojourners who already spent a great part of the year in the Santa Maria Valley to settle there permanently and to subsequently transplant their families from Mexico. Although these recent settlers received authorization to remain in the United States thanks to the Special Agricultural Workers program, the imported dependents have not been authorized. Finally, as indicated above, settled families habitually provide kin with sanctuary during their seasonal sojourn from Mexico to the valley and, hence, add to the growing number of undocumented aliens to be found in their midst. Because many of the undocumented are close kin, immigrant families will not readily or voluntarily reveal their presence to anyone; they are, rather, quite determined to shield them from detection and possible deportation. It is necessary to note that immigrants’ dogged determination to conceal undocumented relatives, even from innocuous surveyors, increases exponentially as the anti-immigrant sentiment we have witnessed in recent times swells. Local, state, and federal “get tough with immigrants” measures which, among other results, propose to bar children from school, deprive workers from access to basic health services, and rescind citizenship from the children of undocumented parents born in the United States are all unmistakable signs that the risk factor of detection is greater than ever. Cautious suspicion, as a result, is heightened to near paranoia when it is rumored that, among others, teachers, doctors, social workers, and “good” citizens at large will be asked, if not required, to report the presence of undocumented aliens to proper government authorities. Finally, because many immigrant families lease parts of their dwellings to non-kin sojourners, violating in the process local housing ordinances and rental agreements, they are not inclined to reveal or report their presence to anyone. Moreover, they can become particularly apprehensive about this matter because boarders provide an income that probably goes unreported to the Internal Revenue Service.Easy to confuse with the growing ranks of settled immigrant families described above are some 3,000 workers who, although they appear to have settled permanently in the Santa Maria Valley, really have not. That is, though they display evidence of settlement by having both consumers and producers living in stable and well-organized domiciles in the valley, they also continue to maintain a principal place of residence in the Mexican home community. Some actually own and maintain two homes, one in Mexico and the other in the Santa Maria Valley. Members of these families move back and forth between the two homes incessantly, some at regular intervals following, for example, farm employment cycles and school schedules, and others seemingly at random.

The specifics of the organism’s capabilities and the environmental features that structure resource selection opportunities are constraints. In the diet breadth model constraints include things like the size of the forager, the hunting and gathering technology used, and the distribution and caloric value of the targeted resources. Constraints are all of the elements of the situation that are taken for granted , in order to focus analysis on one set of effects. The measure we use to assess costs and benefits is known as the currency. While the currency might be any feature of a resource that gives it value, foraging theorists typically assume that food energy is the most important attribute. After oxygen and water, mammals require metabolic energy in large amounts on a nearly continuous basis. The omnivorous diet of most hunter-gatherers makes it likely that meeting one’s need for energy entails meeting the needs for other nutrients. This may be more problematic with agriculturalists. The kcal currency is expressed as an efficiency, the net acquisition rate of energy. Where energy is not limiting or is less limiting than some other factor—e.g., protein—then that can be used as the currency. For instance, we know that some forms of energy, especially those from large or dangerous game animals, are more prestigious than others , suggesting that not all kilocalories are equal. Prestige might enter into the currency in some cases. Behavioral ecologists generally emphasize secondary currencies like kcals or mating success because they are more tractable than the primary neo-Darwinian measure of reproductive fitness . The final feature of models is the goal. A deterministic foraging model likely would have the goal of maximizing energy capture while foraging. A risk-sensitive model would emphasize the goal of avoiding harmful shortfalls of energy. Behavioral ecology models of food transfers in a social group might stress the evolutionarily stable equilibrium of distribution tactics.

The polygyny threshold model for mating tactics would emphasize the goal of reproductive success. Different goals usually imply different methods: simple optimization analysis for energy maximization; stochastic models for risk minimization; game theory for frequency dependent behaviors, like intragroup transfers, best indoor plant pots that result in evolutionarily stable strategies. The optimization assumption ties together constraints, currency, goal, and the costs and benefits of the alternative set. For instance, given constraints of resource densities and values, and their associated costs and benefits, we predict that organisms will select the alternative that provides them the highest available net acquisition rate of energy. As noted earlier, even when there is no particular shortage of foodstuffs, efficient foraging frees time for alternative activities and lessens exposure to risks associated with foraging. While we don’t expect the organism always to engage in the optimal behavior, models based on this assumption have proven to be robust when compared to ethnographic and archaeological datasets .The diet breadth or resource selection model is one of the oldest and most commonly used , particularly by archaeologists . It is sometimes called the encounter contingent model because it focuses on the decision to pursue or not to pursue, to harvest or not harvest, a resource once it is encountered. The decision entails an immediate opportunity cost comparison: pursue the encountered resource, or continue searching with the expectation of locating more valuable resources to pursue. If the net return to is greater than , even after allowing for additional search time, then the optimizing forager will elect to pass by the encountered resource, and will continue to do so no matter how frequently this type of resource is encountered. The general solution to this trade-off is devised as follows: each of k potential resources is ranked in descending order by its net return rate for the post-encounter work to obtain it. This represents a resource’s net profitability with respect to pursuit, harvest, and handling costs.

The derivation of the best-choice diet begins with the most profitable resource , and, stepwise, adds resource types, continuing until the first resource with a profitability less than the overall foraging efficiency of the diet that does not include it . Resources ranked are excluded because to pursue them would impose an unacceptable opportunity cost: a lower return rate for time spent pursuing them relative to the expected benefits from ignoring them in favor of both searching for and pursuing more profitable types. Think of picking up change in tall grass: if there are enough silver dollars and quarters the income-minded gleaner will ignore the dimes, nickles, and pennies, no matter how frequently they are encountered. Notice that the DBM also entails a marginal decision: It asks, is the profitability of the next ranked item above or below the marginal value of foraging for all resources ranked above it? Creative use of this or any foraging model entails thought experiments of the form: how will an optimizing forager respond to a change in independent variable x. Predicted responses are confined to options with the alternative set, but the independent variable x might be any change in the environment or the behavioral capacities of the forager that affects the primary model variables: resource encounter rates and profitability. For instance, resource depression,environmental change, and other factors which diminish encounter rates with highly ranked resources will increase search costs, lower overall foraging efficiency, and as a result, may cause the diet breadth of a forager to expand to include items of lower rank. One or more items that previously ranked below that boundary may now lie above it, making these resources worth pursing when encountered. The converse is also true. Sufficiently large increases in the density of highly ranked resources should lead to exclusion from the diet of low ranked items. A seasonal elevation of fat content, or adoption of a technology that makes its pursuit, harvest or processing more efficient or any factor that raises the profitability of a particular resource will elevate its ranking, perhaps enough to move into the best-choice diet. It may, in fact, displace resource items previously consumed. Winterhalder and Goland provide an extended list of factors that might operate through encounter rate and pursuit and handling costs to change resource selectivity. The diet breadth model also implies that, under a given set of conditions, resources within the optimal diet are always pursued when encountered; those outside the optimal diet will always be ignored. There are no “partial preferences,” such as “take this organism 50% of the time it is encountered.” Likewise, the decision to include a lower-ranked item is not based on its abundance, but on the abundances of resources of higher rank. Think of the small change mentioned earlier.In the diet breadth model, we envision a resource that is harvested as a unit with a fixed value .

By contrast, a patch is a resource or set of resources which is harvested at a diminishing rate, either because it is depleted in such a way that makes continued harvesting more difficult; the densest and ripest berries are picked first, blueberry container size or because the continuing presence of the forager disperses or increases the wariness of remaining resource opportunities as in the second or third shot at a dispersing flock of grouse. Patches can be ranked like resources, by their profitability upon encounter. As a first approximation, the same predictions apply. However, predictions are somewhat less clear for the selection of patches than for resources, because a definitive prediction about patch choice is interdependent with a decision about patch residence time, the focus of the next model.If a resource patch—which we envision as a small area of relatively homogeneous resource opportunities, separated by some travel distance from other such locales—is harvested at a diminishing rate of return, it is obvious to ask when the forager should abandon its efforts and attempt to find a fresh opportunity. By moving on, he or she will incur the cost of finding a new patch, but upon locating it, will be rewarded with a higher rate of return, at least for a while. The marginal value theorem postulates a decline in return rates for time spent in the patch, usually approximated by a negative exponential curve. The optimizing solution specifies that the forager will leave the present patch when the rate of return there has dropped to the average foraging rate. The average foraging rate encompasses the full set of patches being harvested and the travel costs associated with movement among them. To stay longer incurs unfavorable opportunity costs because higher returns were available elsewhere. To stay a shorter duration is also sub-optimal, because rates of return are, on average, higher when compared to the costs of moving on to another resource patch. In this model, short travel times are associated with short patch residence ; long travel times with longer residence times. The forager optimizing his or her patch residence time rarely will completely deplete a patch;the resources left behind are significant for the recovery of the patch. Finally, the value of harvested patches, upon departure, is the same. The inter-dependence between the two patch related models should now be more apparent. Predictions about patch residence time depend on patch choice; reciprocally, predictions about patch choice depend on residence time. Use of one of these models must assume the other; Stephens and Krebs give a more detailed discussion of this model.The ideal free distribution is a model of habitat choice . The distinction between patches and habitats is one of scale: patches are isolated areas of homogenous resource opportunities on a scale such that a forager may encounter several to several dozen in a daily foraging expedition. Habitats are similarly defined by their aggregate resource base, but at a regional scale. As suggested by their greater relative size, habitats also invoke somewhat different questions, such as where to establish and when to move settlements, and when to relocate by migration. Generally, we ask how populations will distribute themselves with respect to major landscape features like habitats. In the ideal free distribution, the quality of a habitat depends on resource abundance and the density of the population inhabiting and using it. The model assumes that the initial settlers pick the best habitat, say “A.” Further immigration and population growth in habitat A reduce the availability of resources and the quality of the habitat drops for everyone. Crowding, depletion of resources, and competition are possible reasons for this. The marginal quality of habitat A eventually will drop to that of the second-ranked, but yet unsettled, habitat B. If each individual in the population seeks the best habitat opportunity, further growth or immigration will be apportioned between habitats A and B such that their marginal value to residents is equalized. Lower ranked habitats will be occupied in a similar manner. This model predicts that habitats will be occupied in their rank order, that human densities at equilibrium will be proportional to the natural quality of their resources, and that the suitability of all occupied habitats will be the same at equilibrium. In the IFD the creative element resides in imaging how various socioenvironmental settings might affect the shape of the curves representing the impact of settlement density on habitat quality. For instance, it is possible that settlement at low densities actually increases the suitability of a habitat. Forest clearing by the newcomers leading to secondary growth might increase the density of game available to them and to emigrants. This is known as the Allee effect. Likewise, some habitats may be quickly affected by settlement, generating a sharply declining curve of suitability as population densities increase, whereas others may be much more resilient. If immigrants to a habitat successfully defend a territory there, then newly arriving individuals will more quickly be displaced to lower ranked habitats, a variant known as the ideal despotic distribution .Many foragers, human and nonhuman, locate at a dry rock shelter, potable water, or a valuable or dense food source or other particularly critical resource—e.g., an attractive habitation site, or perhaps at a location central to a dispersed array of required resources—and then forage in a radial pattern from that site. Central place foraging models address this circumstance. They assume that a forager leaving such a home base must travel a certain distance through unproductive habitat to reach productive foraging zones.

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 .