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As pointed out in Ref., the Berry phase has a profound geometrical origin because an adiabatic and cyclic process of a quantum state is mathematically equivalent to parallel transporting it along a loop, which connects to the concept of holonomy in geometry. Hence, the Berry phase bridges physics and geometry, making it extremely important in the understanding of topological phenomena, such as integer quantum Hall effect, topological insulators and superconductors, and others. The description of the Berry phase relies on the properties of a pure state of a quantum systems at zero temperature. Meanwhile, mixed quantum states, including thermal state at finite temperatures, are more common. Therefore, mixed-state generalizations of the Berry phase have been an important task. Uhlmann made a breakthrough by constructing the Uhlmann connection for exploring the topology of finite-temperature systems. As the Berry holonomy arises from parallel transport of a state-vector along a closed path, the Uhlmann holonomy is generated by parallel-transporting the amplitude of a density matrix. defined by W = √ρU. Here the amplitude W is the mixed-state counterpart of the wavefunction, and U is a phase factor. A geometrical phase is deduced from the initial and final amplitudes. However, Uhlmann’s definition of parallel transport is rather abstract and may involve non-unitary processes, complicating a direct and clear physical interpretation. Moreover, the fiber bundle built upon Uhlmann’s formalism is trivial, which severely restricts its applications in physical systems.Purification of a mixed state leads to purified state, round flower buckets a state-vector equivalent to the amplitude of a density matrix. The lack of a one-to-one correspondence between the density matrix and its purified states gives rise to a phase factor, similar to the phase of a wave function.

In a branch of quantum field theory called thermal field theory, there is a similar structure for describing the thermal-equilibrium state of a system by constructing the corresponding thermal vacuum by duplicating the system state as an ancilla and forming a composite state. It plays a crucial role in the formalism of traversable wormholes induced by the holographic correspondence between a quantum field theory and a gravitational theory of one higher dimensions. Importantly, purified states of a two level system has been demonstrated on the IBM quantum computer while the thermal vacuum of a transverse field Ising model in its approximate form has been realized on a trapped-ion quantum computer. Despite the superficial similarity, a major difference between a thermal vacuum and a purified state is a partial transposition of the ancilla to ensure the Hilbert-Schmidt product is well defined. In quantum information theory, a partial transposition is closely related to entanglement of mixed states. Importantly, partial transpositions of composite systems have been approximately realized in experiments by utilizing structural physical approximations in suitable quantum computing platforms. Although ordinary observables cannot discern the partial transposition between the purified state and thermal vacuum, here we will show that at least two types of generalizations of the Berry phase to mixed states are capable of differentiating the two representations of finite temperature systems. Among many attempts to generalize the Berry phase or related geometric concepts to mixed states , a frequently mentioned approach was proposed in Ref.. Instead of decomposing the density matrix to obtain a matrix-valued phase factor, a geometrical phase is di-rectly assigned to a mixed state after parallel transport by an analogue of the optical process of the MachZehnder interferometer. Hence, the geometrical phase generated in this way is often referred to as the interferometric phase. The interferometric phase has been generalized to nonunitary processes, but the transformations are still on the system only. Moreover, it is essentially different from Uhlmann’s theory since the conceptual structure of holonomy is not incorporated.

We will first derive a mixed-state generalization of the parallel-transport condition for generalizing the Berry phase without invoking holonomy. This approach unifies the necessary condition for both the interferometric phase and Uhlmann phase . Two ways to implement the parallel-transport condition based on how the system of interest undergoes adiabatic evolution will be introduced, and they lead to different generalizations of the Berry phase. We will name one thermal Berry phase and the other generalized Berry phase. Importantly, the partial transposition of the ancilla between a purified state and thermal vacuum will be shown to produces observable geometrical effects in both thermal Berry phase and generalized Berry phase. Through explicit examples, the two generalized phases are shown to differentiate the two finite-temperature representations, a task beyond the capability of the conventional interferometric phase or Uhlmann phase. The rest of the paper is organized as follows. Sec. II summarizes the Berry phase in a geometrical framework with an introduction of the parallel-transport condition for pure quantum states. In Sec. III, we review the representations of mixed states via purified states and thermal vacua and then explain the difference of the partial transposition of the ancilla. In Sec. IV, we introduce the thermal Berry phase via generalized adiabatic processes. While the thermal Berry phase can differentiate a purified state from a thermal vacuum, it may contain non-geometrical contributions. In Sec. V, we generalize the parallel-transport condition to involve the system and ancilla and derive the general Berry phase according to the generalized condition. While the generalized Berry phase only carries geometrical information, its ability of differentiating a purified state from a thermal vacuum depends on the setup and protocol. We present examples of the thermal and generalized Berry phases. Sec. VI concludes our study. Some details and derivations are given in the Appendix.While purified states of a two-level system incorporating environmental effects have been simulated on the IBM quantum platform, thermal vacua of the transverse Ising model has been experimentally realized on an ion-trap quantum computer by the quantum approximate optimization algorithm. Moreover, partial transposition of a composite system has been approximately realized on quantum computers with various numbers of qubits. Therefore, a comparison of the geometric effects reflected by the generalizations of the Berry phase of purified states or thermal vacua is expected to be achievable in future experiments on quantum computers or quantum simulators. For example, one may consider two identical composite quantum systems of Example V.1 of the generalized Berry phase and then apply a partial transposition to one of the composite systems. As a consequence, the composite system with a partial transposition corresponds to a purified state while the one without partial transposition may be viewed as a thermal vacuum. By applying parallel transport that involves the ancilla to both composite systems and extract their generalized Berry phase after a cycle, a π-phase difference is expected between the two composite systems. Given the large phase difference between them after a cycle, the result is robust against small perturbations or noise from the hardware and offers another demonstration of geometrical protection of information. We have presented two generalizations of the Berry phase, the thermal Berry phase and generalized Berry phase, for distinguishing the two state-vector representations of mixed states via the purified state and thermal vacuum. From the geometrical and physical points of view, the generalized Berry phase has more desirable properties since the thermal Berry phase is generated by a temperature-dependent thermal Hamiltonian and may carry non-geometrical information. We caution that while the transformations can be on the system, ancilla, or both in the construction of the generalized Berry phase, an operation on the ancilla is necessary if we want to differentiate the purified state and thermal vacuum.The earliest classification of the forms of matter we see around us, typically presented to us in our early school days, consists of solids, liquids and gases.

High school physics textbooks and experience later teach us that solids can be further classified based on their electronic properties as conductors and insulators. Solid state physics courses in college add semiconductors, semimetals and superconductors to that list, plastic flower buckets wholesale and explain the basic physics that governs the electronic properties of these phases. More precisely, as long as the electrons in a solid are non-interacting, solids with partially filled bands are shown to be metals or conductors while those with no partially filled bands and a gap between the valence and the conduction bands are insulators or semiconductors. If the gap is extremely small or vanishing or if there is a very small overlap between the valence and the conduction bands, the material is semimetallic. Superconductors are argued to be the fate of a metal cooled to extremely low temperatures. However, vast quantitative as well as qualitative differences between the properties of materials within a single category are often observed. For instance, some insulators have conducting surfaces while others do not. Moreover, the surface conduction is stable against perturbations as well as deformations in the band structure as long as the underlying symmetries of the system are preserved and the system remains insulating after the deformations. Similarly, interactions and certain perturbations can gap out some semimetals and turn them insulating but not others . These observations call for further refinement in the classification of solids, especially one that explains why the properties of some phases are robust to certain deformations, interactions and perturbations. In other words, an understanding for why some phases are topological while other are not, is required. The study of the topology of the bands provides a powerful unifying framework for accomplishing this task. Topology shows up in two distinct ways in the band structures of non-interacting Hamiltonians depending on whether the spectrum is gapped or gapless. In systems with gapped band structures, which include insulators and most superconductors, the wavefunctions wind non-trivially across the Brillouin zone in the topological phases, as described in Sec. 1.1. What constitutes a non-trivial winding depends strongly on the symmetries and the dimensionality of the system under consideration, thus, revealing a rich substrucutre within the insulating and superconducting phases. On the other hand, gapless band structures contain topological objects in momentum space which can be characterized by the winding of theGreen’s function around the object, as exemplified in Sec. 1.2. A key feature of topological media is that they typically have unconventional surface states which, in many cases, cannot exist independently of the bulk phase in one lower dimension. For instance, the two dimensional surface of a three dimensional topological insulator hosts pseudorelativistic electrons and cannot exist as an independent two dimensional system. Because of this intimate surface-bulk connection and because the surface is usually more accessible than the bulk, experiments usually probe the surface states in order to identify the bulk topological phase. The surface states are also extremely valuable from a practical point of view, since the unconventional properties bestowed upon them by the non-trivial bulk topology may be exploited to design novel electronic devices.The role topology plays in the band structures of gapped non-interacting Hamiltonians is analogous to its manifestation in an early example of topology in physics – Gauss’s law in electrostatics. Gauss’s law states that the total electric flux piercing a closed surface is determined only by the charge enclosed by it and is independent of its shape or the precise charge distribution. Thus, the total flux through each surface in Figure 1.1.1 is four units. For gapped noninteracting lattice Hamiltonians, the analog of the Gaussian surface is the Brillouin zone, and different Gaussian surfaces correspond to different Brillouin zones in the extended zone scheme or to different bands in the reduced zone scheme. The electric charge, then, maps to an appropriate topological invariant whose exact form depends on the symmetries of the system under consideration. This topological invariant can be written as an integral over the Brillouin zone of an appropriate field derived from the Bloch functions of the occupied bands, analogous to how the electric charge enclosed by a Gaussian surface is equal to an integral of the electric field over it. The analogy is generalizable to continuum Hamiltonians, defined over all of momentum space, as well, provided the points at infinity are identified. This identification compactifies momentum space to the topological equivalent of a sphere, which, being a closed surface, permits the application of Gauss’s law. Next, assuming every charge that exists in the universe is inside a Gaussian surface, the only way to change the amount of charge enclosed by a single surface is by fusing it with another Gaussian surface, moving charges across the junction and pinching the junction ofto get the original surfaces back, as illustrated in Figure 1.1.2. Similarly, for band structures, topological invariants can only be defined for Brillouin zones wave functions in the extended zone scheme or equivalently, for bands in the more common reduced zone scheme.

Higher synthesis coupled with reduced transporter activity may act to elevate synaptic dopamine concentrations in older adults, potentially reflecting compensatory mechanisms for counteracting losses in receptor density. However, research to date has not supported a clear role of elevated synthesis in benefiting cognition . Indeed, inverted-U-shaped relationships are observed for dopamine synthesis , suggesting higher synthesis levels may, in some cases, be detrimental. Therefore, it may not be sufficient to consider aging as simply a hypodopaminergic state, but instead may be a dysregulated state characterized by a loss of balance between presynaptic and postsynaptic components of the system. Similar conceptualizations of dopamine dysregulation may be applied to psychological and psychiatric disorders including schizophrenia, ADHD, and addiction . This dysregulation may critically affect the precision of dopamine signaling, increasing the variability of its temporal dynamics and noise . There is emerging evidence that regulation of dopamine receptors and age-related decline in receptor density is not spatially homogeneous . Rieckmann and colleagues report age-related reductions in interregional correlations in D1 BPND using [11C]SCH23390. Their findings suggested that aging is associated with a dissociation in D1 receptor regulation between nigrostriatal and mesocortical/mesolimbic pathways. Seaman and colleagues evaluated regional differences in estimated rates of percentage of change in D2/3 BPND using [18F]fally pride . Their findings revealed tremendous heterogeneity across regions in the estimated rate of decline, which was variably linear or curvilinear. Even after correcting for differences in gray matter volume, procona system they estimated the most extreme reductions in subgenual frontal cortex and superior temporal gyrus and less pronounced reductions in ventral striatum, pallidum and hippocampus .

Altogether, findings in aging indicate that changes in the dopamine system vary across individuals, vary across different presynaptic and postsynaptic components, and vary spatially across the brain. In the following three sections, we highlight instances in which accounting for these three sources of variability may shed light on reported effects in valuebased decision-making.Loss of dopamine has been linked with propensity to avoid punishment rather than approach reward. The probabilistic selection task is a well-known decision-making task that has been used to study these questions and has been applied to healthy adults and a variety of patient populations including those with Parkinson’s disease and schizophrenia . It ostensibly taps into processes of modelfree reinforcement learning, with some evidence suggesting it is preferentially associated with striatal rather than PFC dopamine function . Despite links to dopamine, there have been largely null findings in previous studies examining consistent group-wise effects of age on performance. Here we provide background on the PST, and models of dopamine’s role via the D1-mediated “direct” and D2-mediated“indirect” pathways. We propose that any measure that captures individual differences in dopamine function may useful in clarifying these null effects. Further, we discuss specific ways in which D1 and D2/3 receptor imaging can be leveraged to contribute to our basic understanding of the role of these pathways in human decision-making as well as individual differences in performance in aging. The PST is composed of both a probabilistic learning phase and a choice phase. Its design aims to assess potential biases in choice action to approach reward versus choice action to avoid punishment.

Briefly, three sets of Japanese hiragana characters are presented in randomized order and are associated with differing probabilities of reward or punishment . During the learning phase, participants learn to select the stimuli associated with reward. However, this learning could be driven by positive reinforcement from reward or negative reinforcement from punishment . The choice phase of the PST is designed to dissociate these possibilities. The stimuli are presented in novel pairs to reveal underlying choice biases in incentive motivation to either approach reward or avoid punishment . The bias to avoid punishment was demonstrated in Parkinson’s patients tested off medication, but was reversed when patients were tested on medication in presumably dopamine-replete states . Since the original description of the PST task, there has been significant interest in understanding how age-related changes in dopamine function may affect biases in decision-making. A simple hypodopaminergic account of aging would predict that age effects mimic those observed in Parkinson’s disease, but to a lesser degree given the relative sparing of dopaminergic function . Such biases, if they produce inoptimal choice behavior, would be a prime target for intervention in aging. Behavioral evidence of age-related biases in choice selection is mixed, with greater individual differences reported for older adults. A bias to avoid punishment has been shown in an older subset of older adults, but not in a younger subset . Some studies report greater individual differences in the balance between positively and negatively motivated choices in aging , or have shown selective reduction in positive learning but not negative learning . Other aging studies have found no effects of valence . We posit that accounting for individual differences in the decline of dopamine function using specific dopamine targets in striatum or neuromelanin-sensitive MR will clarify these null and mixed results. Previous research using MR approaches for assessing midbrain dopaminergic nuclei suggest a benefit of greater structural integrity for reward learning .

We predict that the subgroup of older adults with a positive choice bias to approach reward will have greater midbrain neuromelanin MR signal than older adults with a negative choice bias .The proposed mechanisms of dopamine’s involvement in approaching reward versus avoiding punishment involve the weighting of two circuits, the direct pathway versus the indirect pathway, involving substantia nigra, striatum, globus pallidus, and thalamus. Activation of the direct “go” pathway leads to the disinhibition of thalamus, facilitating outputs to cortex. Conversely, activation of the indirect “no-go” pathway suppresses thalamic output to cortex. The direct pathway is associated with reward-based learning and approach, which may be mediated by stimulation of striatal D1 expressing medium spiny neurons . The indirect pathway is associated with aversion-based learning and avoidance, which may be mediated by stimulation of striatal D2 expressing medium spiny neurons . Frank and colleagues suggest that low tonic dopamine amplifies learning through D2 negative reinforcement mechanisms and accounts for biases to avoid punishment in Parkinson’s patients tested off medication . A recent PETstudy in healthy young adults directly probed relationships between individual differences in D1 and D2/3 BPND and propensity to approach reward versus avoid punishment in the PST . However, higher D2/3 BPND was not clearly related to a bias to avoid punishment in this young adult sample. It is currently not known whether D2/3 effects would emerge if these healthy subjects were tested in a dopamine depleted state . It is possible that individual differences in relative ratios of D1 and D2/3 receptor densities may underlie between-subject variability in PST performance in older adults, or variability in performance on other tasks, such as tasks involving risky gambles, which tap into approach versus avoidance mechanisms. There is some evidence, though limited, suggesting D1 and D2/3 receptor densities decline at different rates across the lifespan . Estimated rates of decline are numerically greater for D1 receptors than D2/3 receptors for between-subject comparisons of PET data and within-subject analyses of postmortem tissue . However, additional research is needed to establish whether there are asymmetric effects of aging on D1 versus D2/3 receptors. It is unclear what the underlying physiological mechanism might be for the relative vulnerability of D1 receptors or relative resilience of D2/3 receptors in aging. D1 and D2-expressing medium spiny neurons have distinct morphological and electrophysiological properties which may confer unique susceptibilities. In culture, D2, rather than D1 receptors, procona valencia buckets may be more vulnerable to excitotoxic insults . However, observations in early Parkinson’s disease reveal upregulation of D2/3 receptors, but not D1 receptors . This leaves open the possibility that differences in the capacity for receptor upregulation may underlie reductions of the ratio between D1 and D2/3 in healthy aging. Regardless of mechanism, the direction of these effects observed in aging is generally consistent with the view that aging shifts choice behavior toward a bias to avoid punishment rather than approach reward.There is, however, significant inter individual variability in the trajectory of age-related changes in the dopamine system, which would warrant examination of within-subject ratios of D1 to D2/3 receptor densities and their relationship with performance. For the PST, evaluation of individual subject performance has revealed subgroups of older adults with a “positive” choice bias to approach reward and subgroups with a “negative” bias to avoid punishment . Subjects with losses in D1 receptors but relative preservation of D2/3 receptors may show reduced choice behavior to approach reward , and greater choice behavior to avoid punishment . Subjects with losses in D2/3 receptors but relative preservation of D1 receptors could be expected to show the opposite pattern of results .

In vivo PET imaging could resolve the underlying neural basis of these individual differences in positively and negatively motivated choices in aging. Further, complementary studies in animal models could test whether selective knock-down of D1 versus D2 receptors generates a similar pattern of results.While the density of dopamine receptors declines with age, there is evidence for counteracting increases in dopamine synthesis and decreases in dopamine reuptake via reduced transporter BPND . If imbalance in the tuning of pre and postsynaptic components occurs in aging, this dysregulation may lead to reduced precision of RPEs implicated in value-based reinforcement learning . Such dysregulation would be expected to result in slower model-free reinforcement learning. Behavioral evidence suggests that while older adults perform comparably to young adults when cue reward contingencies are deterministic older adults show impaired performance in situations in which outcomes are probabilistic or require learning from feedback . Though behavioral evidence indicates impaired reward-based learning in older adults, it is not clear that neural activity associated with reward anticipation or reward outcome is systematically altered in aging. Ventral striatum/nucleus accumbens activation in response to reward-predicting cues is the same in young and older adults . Further, responses to rewarding outcomes in ventral striatum and medial PFC have been shown to be similar in young and older adults . These measures of BOLD activation suggest there are no systematic differences in reward responsivity in aging. However, these measures typically rely on averages across many trials, and may not capture age-differences in trial-to-trial variability. Fruitful lines of research in aging have linked reward-based learning with neurocomputational approaches to examine age differences in RPE-like BOLD signal that rely on trial-based estimates. Together, these studies suggest that aging reduces correlations between RPEs derived from reward learning tasks and BOLD activation in ventral striatum/ nucleus accumbens and ventromedial PFC . Few studies have linked age-related reductions in these correlations with alteration in dopamine function in aging. In one notable exception, Chowdhury, Guitart-Masip, Lambert, Dayan, et al. pharmacologically manipulated dopamine to examine its effects on learning and RPEs. This study demonstrated that treatment of older adults with levodopa increased both RPE-like signals in ventral striatum and rates of learning. A recent study using the same task probed relationships between striatal D1 BPND and nucleus accumbens RPE’s in young and older adults . Surprisingly, they did not find striatal RPE-like responses in either young or older adults. D1 BPND was not correlated with performance for either group but was positively related to ventromedial PFC signal associated with reward anticipation. Moving forward, it will be valuable to consider how dopamine changes in aging may fundamentally alter the reliability in dopamine signaling to affect reward-based learning. This can be achieved in studies in animal models that examine the effects of age on the amplitude and timing of phasic responses. In humans, foundational studies could investigate whether neuromelanin-sensitive MR measures of midbrain dopamine function are related to the strength of correlations between RPEs derived from reward learning tasks and striatal BOLD signal in older adults. Higher correlations may be predicted in older adults with higher midbrain neuromelanin MR contrast-to-noise ratios. Applying PET methods, future studies could consider relationships between presynaptic and postsynaptic components within subject. For example, individual differences in the ratio of dopamine transporter availability and D2/3 receptor availability within subject may correlate with rates of learning . One may predict an inverted-U-shaped relationship between a presynaptic/ postsynaptic composite measure and learning suggesting that an optimal balance in dopamine receptor binding and reuptake is associated with more precise RPEs and more efficient learning rates in aging.

Fixed nitrogen is often a limiting nutrient for primary productivity in the surface ocean, and consequently influences the dynamics of oceanic carbon sequestration . Nitrogen fixation by marine cyanobacteria is an important source of oceanic fixed nitrogen, adding an estimated 100–200 Tg-N annually to open ocean ecosystems . This nitrogen fixation is often associated with cyanobacterial trichomes or aggregates colonized by heterotrophic bacteria, picoeukaryotes and metazoans . Respiratory activity within these so-called ‘pseudobenthic’ environments can create ephemeral suboxic to anoxic zones, establishing a niche for facultative anaerobes within otherwise oxygenated surface waters . Emerging evidence suggests that denitrification occurs within these anoxic habitats, coupling processes of nitrogen-fixation and loss at the microscale . While initial studies of marine biological nitrogen fixation focused on colonial filamentous Trichodesmium species and symbiotic, heterocystous Richelia species , more recent work has demonstrated the importance of unicellular diazotrophic cyanobacteria from the order Chroococcales . Diazotrophic UCYN have been studied extensively in the global oceans by surveys of the nitrogenase gene nifH diversity, blueberry in pot which revealed three phylogenetically distinct clades . UCYN-A are small , metabolically streamlined, uncultured cyanobacteria that lack the oxygen-producing photosystem II and live as endosymbionts within haptophytes, a lineage of eukaryotic algae .

UCYN clades B and C are larger , free-living cyanobacteria and include cultured representatives, such as Crocosphaera watsonii and Cyanothece sp. ATCC51142. Studies of aggregate-associated nitrogen fixation have focused predominantly on Trichodesmium sp. colonies and rafts , or filamentous heterocystous cyanobacterial colonies . However, some Crocosphaera watsonii strains have been observed to produce copious quantities of exopolysaccharides and have been linked to the formation of transparent exopolymer particles . These gel-like particles provide microhabitats for other microorganisms, and thus have the potential to play an important role in marine biogeochemical cycling . Here, we report a new species of uncultured, unicellular cyanobacteria from the order Chroococcales which forms millimeter-sized aggregates together with diatoms and other putatively heterotrophic bacteria. These macroscopic aggregates, which we call “green berries,” are found in the muddy, intertidal pools of Little and Great Sippewissett salt marshes . They are found interspersed with previously described, sulfur-cycling “pink berry” consortia . Using a combination of metagenomic sequencing and ecophysiological measurements, we demonstrate that the green berries are characterized by diazotrophy and rapid rates of photosynthesis and respiration that produce steep oxygen gradients. Heterotrophic bacteria within the green berries are closely related to other marine epiphytic marine strains and encode key genes in the denitrification pathway.

The green berries are found in the same organic-rich, intertidal pools of Little Sippewissett salt marsh on Cape Cod where both multicellular magnetotactic bacteria and pink berries have been previously studied . Though less abundant than the pink berries found in these pools , the green berries form similar irregular ellipsoid aggregates measuring 1–8 mm in diameter, with an average equivalent spherical diameter of 1.7 mm ± 0.1 mm . Green berries were dense and compact aggregates that were typically observed at the sediment-water interface, but were occasionally found to float at the water surface when suspended by bubbles. Microscopic observation of the green berries revealed abundant coccoid unicellular cyanobacteria 5–7 µm in diameter , interspersed with pennate diatoms . Filamentous cyanobacteria were observed occasionally, but were rare compared to the unicellular GB-CYN1 morphotype. A clear, extracellular matrix coated these aggregates of phototrophic cells, and was colonized by a variety of smaller bacteria . GB-CYN1 exhibited absorption maxima at 620, 660, and 680 nm corresponding to thepresence of phycocyanin, allophycocyanin and chlorophyll a, respectively.Sequencing of 18S rRNA genes from the green berries indicated that the eukaryotic community was predominantly made up of two different pennate diatom species related to Navicula cari strain AT-82.04c and Amphora pediculus strain AT-117.11 . These same diatom species were also the dominant eukaryotic 18S rRNA gene sequences recovered from pink berry aggregates, though diatoms were more abundant in green berries than in pink berries, as observed by microscopy and the relative abundance of 16S rRNA chloroplast sequences .

Bacterial 16S rRNA gene sequences amplified from the green berries were dominated by sequences related to either diatom chloroplasts or Chroococcales unicellular cyanobacteria . Unassembled metagenomic sequence reads assigned to rRNA sequences and protein-coding regions support the observed abundance of Chroococcales , but did not recover comparable proportions of diatom chloroplasts . FIGURE 2 | Comparison of green berry bacterial diversity estimates from16S rDNA PCR amplified clones library with unassembled Roche 454 metagenomic sequence reads. Taxonomic assignment of metagenomic reads matching ribosomal RNA reads was conducted using the M5RNA database in MG-RAST . A similar taxonomic assignment was conducted with metagenomic reads matching protein coding sequences in the M5NR database . Note that the 16S rRNA clone library abundance data for the Bacillariophyta was obtained from diatom chloroplasts sequences, which are likely present in multiple copies in the cell and thus not directly comparable to metagenomic 18S rRNA sequences for this group . The overall bacterial community structure of the green berries was significantly different from coexisting pink berry consortia . Some abundant taxa from the pink berries co-occurred in the green berries as rare OTUs, such as the purple sulfur bacterial species Thiohalocapsa sp. PB-PSB1 , and a Winogradskyella species . The persistence of these distinct, co-occurring pink and green berry consortia suggests that the process of macroscopic aggregation enables niche partitioning between oxygenic and anoxygenic phototrophs in these marsh pools. Most of the non-cyanobacterial sequences in the green berry consortia are related to aerobic and facultatively anaerobic marine heterotrophs from the Bacteroidetes, Alphaproteobacteria, and Gammaproteobacteria . Many of these sequences were most closely related to environmental 16S rRNA sequences associated with aggregates of oxygenic phototrophs. Examples of such habitats included phytodetrital aggregates collected from euphotic and hadal environments , and epiphytes of marine macroalgae . The occurrence of related phylotypes in such environments suggests that taxa may be well adapted to an attached lifestyle, degradation of photosynthate, and the fluctuating oxygen conditions in an aggregate environment.Metagenomic data indicate that the orders Rhizobiales and Rhodobacterales of the Alphaproteobacteria are abundant in the green berry consortia. While these groups were rarely detected in the PCR-based 16S rRNA survey, we have previously observed this same PCR bias from the 8F primer during studies of the pink berry consortia . We find the abundance of these clades in the green berries particularly interesting as they include lineages of marine denitrifying bacteria. For example, pelagic Rhizobiales have been linked to denitrification when found in association with macroscopic Trichodesmium sp. aggregates in oxic waters bordering oxygen minimum zones .The cyanobacterial 16S rRNA gene sequences from the green berries grouped into two closely related OTUs , GB-CYN1a and GB-CYN1b, that can be confidently placed in the order Chroococcales . The GB-CYN1 monophyletic cluster formed a clade basal to the UCYNA clade . Using 29 concatenated single-copy phylogenetic marker genes assembled from the metagenome , plastic planters wholesale we reconstructed a phylogenetic tree that placed the GB-CYN1 within a clade including Crocosphaera watsonii and Cyanothece sp. ATCC 51142 as a sister taxa to “Candidatus Atelocyanobacterium thalassa” isolate ALOHA . A phylogenetic tree inferred from nifH gene sequences reveals that the near full-length nifH gene recovered from the GB-CYN1 metagenomic data affiliated with the UCYN-B clade, and was most closely related to Cyanothece sp. 8801/8802 and Crocosphaera watsonii . We conclude that the observed discordance between 16S rRNA, concatenated, and nifH gene phylogenies involving species such as Cyanothece sp. 8801, Gloeothece sp. KO68DGA, and the cyanobacterial endosymbiont of Rhopalodia gibba is most likely due to lateral gene transfer of the nifH gene. Lateral transfer of nifH has been observed in many other species, including mat-forming filamentous cyanobacteria .A full suite of nitrogenase genes were found in the green berry metagenome and were consistently assigned to GB-CYN1, indicating the metabolic potential for nitrogen fixation characteristic of other members of the UCYN A-C clades . In two separate experiments , we measured rates of 11 and 20 nanomoles acetylene reduced per hour per milligram of aggregate dry weight . These rates are comparable, though faster than the rates of 3–6 nmol mg−1 hr−1 reported in macroscopic aggregates of filamentous cyanobacteria from Bogue Sound, North Carolina . Rates measured from actively growing Cyanothece cultures were two orders of magnitude larger than the rates in the green berries.

The total aggregate elemental composition was analyzed using elemental analyzer isotope ratio mass spectrometry . The mean observed C:N ratio in the green berries, 7.1 ± 0.6 , falls within range of the Redfield molar ratio . This observed C:N ratio is higher than that the ratio of 5.4 ± 0.4 observed in similarly large, anoxic, diazotrophic Nodularia spumigena aggregates from the Baltic Sea . Cultures of Crocosphaera watsonii exhibit wide diel fluctuations in C:N ratios as a result of temporal partitioning of carbon and nitrogen fixation activities . Our samples, collected in the late afternoon on a 14 h light/10 h dark photoperiod, are comparable to reports of C:N = 7 from C. wastsonii at similar late afternoon times in a 16 h light/8 h dark photoperiod . Future studies investigating the temporal partitioning for such activities in the green berry aggregate would be informative to elucidate the dynamics of carbon and nitrogen flow in the consortia.Oxygen microsensors were used to characterize the balance between respiration and photosynthesis in the green berry aggregates. Aggregates examined were relatively symmetric ellipsoids of similar size with an equivalent spherical diameter of 1.7 ± 0.1 mm . Photosynthesis produced supersaturated oxygen concentrations within the aggregates: 380 µM O2 with illumination at 170 µE m−2 s −1 and 520 µM O2 at 320 µE m−2 s −1 . During these experiments, bubbles were not observed on aggregate surfaces and the aggregates never floated. Oxygen production rates per aggregate were calculated from these profiles as 13 and 31 nmol O2 per hour at 170 µE m−2 s −1 and 320 µE m−2 s −1 , respectively . We did not rigorously determine the saturating light intensity; however, we observed that illumination with a third lamp failed to stimulate increased oxygen production beyond that with two lamps , suggesting that the saturating light intensity lies in the range of 170 – 320 µE m−2 s −1 . This rate, equivalent to a volumetric rate of 6.8 µmol cm−3 hr−1 , is well above the theoretical threshold for respiration rates capable of causing oxygen diffusional limitation from surrounding seawater . In light-dark shifts, we observed a rapid response where the aggregate core transitioned from steady state supersaturation to full anoxia in 5–8 min . Volumetric gross photosynthetic rates were calculated by two methods: from depth microprofiles via the sum of net photosynthesis and dark respiration and via the light-dark shift technique performed at a single point in the aggregate center . At both light intensities examined, rates calculated via the light-dark shift method were found to be 4.5 µmol cm−3 hr−1 , lower than those from depth microprofiles. While this difference could arise from biological variability between aggregates, we suspect that the light-dark shift rates measured at the aggregate core were lower than those we might have measured closer to the aggregate surface. Future depth integrated studies of photosynthetic rates will help to clarify this difference and allow better characterization of respiratory activity in the light. Comparing the green berries’ dark respiration and gross photosynthesis to other photosynthetic mats and aggregates, we find them similar to the high rates measured for large , filamentous aggregates of the heterocystous cyanobacterium, Nodularia spumigena from the Baltic Sea . Indeed, our estimates of carbon fixation are close to prediction of 349 ng C per aggregate per hour calculated using Ploug et al.’s regression of volume to gross photosynthesis from a 2009 Nodularia bloom. The green berry dark respiration rate was similar, though slightly higher than that observed for Nodularia aggregates of similar diameter; however, the ratio of dark respiration to gross photosynthesis of 0.38 was identical to that observed for 7 mm3 aggregates found late in the bloom . In large Nodularia aggregates, dark anoxia is associated with active dissimilatory nitrogen cycling, including denitrification, dissimilatory reduction of nitrate to ammonia , and significant rates of cryptic nitrification .

Sections were then transferred to 1.5 ml of 0.15% Proteinase K solution and mixed at 55°C and 300 RPM rotation for 14 days with an Eppendorf Thermomixer . Samples were then washed in DI water and placed into an 0.1% amylase solution for 24 hours at 50°C. Samples were then washed in DI water again, lyophilized overnight, mounted on aluminum stubs, and viewed under a Field Emission Scanning Electron Microscope . Sieve plates were viewed under low vacuum , 20- KV of accelerating voltage and a spot size of 2.5.Phloem anatomy was a stronger predictor of maximum sugar accumulation rates than vine carbon gain or water stress. Maximum sugar accumulation rates were not significantly correlated with photosynthesis or midday leaf water potentials . Including photosynthesis and midday leaf water potential as additional predictors also did not substantively improve the relationships between maximum sugar accumulation rates and petiole or pedicel cross-sectional phloem areas. Akaike Information Criterion corrected for small sample size values were higher for the larger models than the univariate models predicting maximum accumulation rates from petiole or pedicel phloem area alone, indicating that accounting for vine carbon gain and water stress did not improve predictive capacity for sugar accumulation . In addition, only one correlation was found between phloem petiole area and minimum mid-day water potential , while other average photosynthesis and water potential variables did not correlate with the phloem anatomical parameters. Finally, a previous dataset measuring leaf area for each cultivar did not find any significant correlations with °Brix accumulation, or other parameters measured .Overall, collection pot we found that total cross-sectional phloem area in the pedicels and the petioles significantly predicted maximum °Brix accumulation rates in the berries , as well as sieve element area in pedicels .

Other sieve tube traits, such as sieve plate porosity, were not correlated with sugar accumulation rates, indicating that grapevines mainly increase their maximum capacity for sugar transport by adding more and wider sieve tubes to the transport pathway. Total cross-sectional areas were significantly lower in cultivars typically grown in hot than warm growing regions, suggesting these cultivars have been inadvertently selected for smaller phloem areas to slow sugar accumulation, delay ripening, and achieve an optimal flavor profile provided by longer grape maturation times prior to harvest . Further, although there wasn’t a significant difference in sieve element area between cultivar climate category in the pedicel phloem, sieve element area did significantly predict brix accumulation rate. Phloem area was also a stronger predictor for sugar accumulation rates than the typical vegetative physiology parameters of gas exchange and water potential . This study points to a new anatomical phenotype that can be used by grape breeders to select for cultivars with smaller petiole or pedicel phloem areas to decrease sugar accumulation rates to berries as an adaptation to increasing temperature.Our phloem area and °Brix accumulation results align with findings from trait comparisons in other crop species and experiments manipulating phloem area in grape and other crops. In grapevine , abscisic acid and gibberellin hormone treatments increased the phloem cross-sectional area in the midveins, pedicels, and stems along with berry sugar concentrations, despite reduced photosynthetic assimilation . The increased phloem area enhances the hydraulic conductivity of the transport pathway , facilitating the transport of sugars from source to sink .

Phloem area has also been linked to fruit growth and sugar accumulation in other crop species. For example, modifying the expression of a phloem cell proliferation regulatory gene in tomato increased phloem area, yield, and fruit sugar concentration . Similarly, in giant pumpkin varieties, the phloem area in pedicels and petioles was positively correlated with fruit yield . These findings highlight the potential for optimizing phloem area to enhance plant productivity by matching source production and sink utilization. Additionally, our study suggests that targeting phloem/xylem in petioles could be an efficient approach for plant breeders to improve yield by enhancing hydraulic conductance and carbon export to fruits .One of the goals of this study was to investigate how cultivars adapted to different climate regimes varied in sugar accumulation and vascular anatomy traits under common garden conditions. Approximately half of the variance in berry sugar concentration is attributable to climate , making common garden experiments crucial to isolate the effects of plant traits on sugar accumulation. We found that, for red varieties, total phloem cross sectional area in the petioles and pedicels was significantly larger in the varieties typically grown in warm regions than hot regions . This could be an adaptation unknowingly selected by generations of winemakers to slow sugar accumulation and synchronize sugar and flavor development in hot climates. For white varieties, phloem area did not increase significantly from hot to warm regions . There could have been less selective pressure to increase sugar accumulation in the warm-climate white than red varieties, since white wines are typically made with lower alcohol content, and the absence of anthocyanin production could reduce metabolic demands for sugar .

Phloem anatomy is influenced by both the climate that plants have adapted to and the climate plants experience during the growing season . This suggests that more work is needed to evaluate how plastic responses to inter annual or geographic variability to climate influence cultivar differences in phloem anatomy and sugar transport capacity. In Arabidopsis, the effects of growing conditions on phloem anatomy depended strongly on the climate the genotypes evolved in. Comparisons between cool and hot growing conditions showed that high temperatures reduced the proportion of phloem area in the minor veins, and that these reductions were larger in Arabidopsis genotypes that evolved in cool than hot climates . These results suggested that phloem plasticity in response to growing conditions outside evolved temperature ranges was greater ingenotypes adapted to cool climates, increasing genotypic differences in phloem anatomy under hot growing conditions. Interestingly, we found the opposite pattern in grape, that phloem area in the pedicel and petiole was significantly larger in the cultivars typically grown in warm than hot climates, even though our common garden experiment was in a hot growing region . Comparisons in different regions or in years with different climatic conditions are needed to determine how strongly the cultivar differences in anatomy observed here depend on the conditions during phloem development.Xylem and phloem area scaled in the midvein, petiole, and pedicel, which produced similar relationships in xylem and phloem areas with maximum °Brix accumulation rates and climate groupings . The relationships with xylem area could simply reflect selection for phloem traits and developmental constraints that make xylem and phloem differentiation proportional, or both xylem and phloem area could impact °Brix accumulation rates. °Brix is a concentration and determined by water and sugar contents. The phloem supplies most of the water to the berries after veraison . The total volume of phloem water influx is generally much larger than the volume of the berries, forcing the berries to export water to the canopy through the xylem to avoid cracking or splitting . A larger phloem area would increase the water influx into the berries, which could require a larger xylem area to compensate for water export. Further, the xylem accounted for most of the vascular area in each organ, and the ratio of xylem to phloem area increased with stem cross sectional area, which also made this ratio significantly larger in warm- than hot-climate cultivars . This larger xylem:phloem ratio could accelerate °Brix accumulation by increasing the capacity for water export relative to influx. Thus, selecting for a lower xylem:phloem ratio could slowberry sugar accumulation. Xylem and phloem areas also scale in other species, including ash , Pelargonium , fir , poplar, and ginkgo , and, notably, xylem: phloem ratios were smaller in species with larger fruit . However, some grape cultivars produce blockages in the pedicel xylem during ripening that reduce conductivity and water efflux, 10 plastic plant pots which could make the ratio of xylem to phloem area less important to °Brix accumulation rates. Overall, more work is needed to clarify the effects of individual tissue areas and xylem: phloem area ratios on ripening.Another interesting findings from the current study was that sieve element area was a significant predictor of brix accumulation rate , while porosity of the elements , most of the smaller sieve element area/ lower brix accumulation rate cultivars were from the warm climate category. These findings suggest that grapevines have primarily adapted to control sugar accumulation rate by changing the number and width of sieve elements, although these two traits were not correlated . Conversely, phloem cross sectional area and mean sieve element area were correlated in the pedicels for other species, including pumpkin and tomato . However, similar to our findings, variation in sieve element area was small for pumpkin, and the differences between cultivars were not significant .

Cooler growing regions are typically more humid and prone to disease pressure , and less porous sieve plates can facilitate the faster formation of callose blockages to more quickly restrict pathogen spread through the phloem . Future work may consider the transcriptional abundance of sugar unloading proteins , and how this relates with phloem anatomical characteristics related to pathway resistance.Phloem anatomy was a stronger predictor of berry sugar accumulation rates than vegetative physiology parameters capturing vine carbon gain and water status. This was unexpected, since photosynthesis determines the carbon available for ripening, and water stress has been shown to strongly impact sugar accumulation rates in many of the cultivars tested here . However, our experimental vines were irrigated during the ripening period to maintain leaf water potentials in a relatively narrow range . This irrigation regime follows standard commercial practices for California, which could have limited cultivar differences in vine water stress and photosynthesis and thus, their impacts on sugar accumulation. These findings suggest that measuring phloem anatomy could provide more insight into plant capacity for berry sugar accumulation under standard, irrigated conditions than conventional vegetative physiology traits. Alternatively, leaf-level photosynthesis could have been decoupled from °Brix accumulation by variation in vine balance , which would impact the ratio of whole-plant carbon supply to demand. A larger ratio of canopy area to fruit mass would increase maximum °Brix accumulation rates. Future work should estimate leaf area per cultivar to ensure that relationships between phloem anatomy and maximum °Brix accumulation rates scale with variation in vine balance.Overall, we found that phloem cross-sectional area in the petioles and pedicels was the most predictive trait for the maximum rate of sugar accumulation in the berries across winegrape cultivars tested. Carbon dioxide in the atmosphere is expected to double by centuries’ end, and the dual effects on plant carbon availability and growing season temperature are projected to strongly accelerate sugar accumulation and exacerbate the detrimental impacts on wine quality . We suggest that reduced phloem areas could be a useful and novel phenotype to screen for in existing cultivars to slow carbon transport rates in hotter growing regions, allowing more time for flavor development. If petiole phloem area is well-conserved across life stages and under different growing conditions, this would be an especially useful trait to accelerate phenotyping since grapevines must mature for several years to begin producing fruit. However, future work is still needed to clarify how xylem area and vine balance influence sugar concentrations, and how interannual and site-specific environmental variability influence anatomical traits and sugar accumulation.Age-related macular degeneration is the leading cause of blindness among seniors in developed countries, and third worldwide after uncorrected refractive errors and cataracts. In early stages, the disease is characterized by small to intermediate drusen with pigmentary changes that may progress rapidly to more advanced forms such as choroidal neovascularization or central geographic atrophy with loss of central vision. Lutein , zeaxanthin , and the isomer meso-zeaxanthin are macular pigments that filter damaging blue light and provide oxidative defense in the macula. These pigments are found in plants as xanthophylls, with increased dietary intake proposed to reduce the development and progression of AMD. The relative concentration of xanthophyll carotenoids in the retina can be measured non-invasively by psychophysical and objective methods, expressed as macular pigment optical density. Numerous epidemiological studies report that individuals with a low MPOD level are at an increased risk of AMD. Dietary L and Z are found in certain fruits and vegetables with red, yellow, or orange color, egg yolk, and in some green leafy vegetables.

Orienting vineyards N-S on a flat terrain allowed for uniform canopy and cluster exposure to solar radiation, since radiation is approximately symmetric about the N-S axis . However, the berry temperature on the west side of the vine significantly increased in the afternoon compared to the east side because hysteresis in air temperature causes asymmetry about solar noon. On average, berry temperature on the west side of the vines was greater than 35◦C for about 1-2.5 hours longer than the east side . Interestingly, although there was temporal asymmetry due to air temperature and temperature extremes, the net daily accumulation of berry growing degree hours was virtually identical between each side of the vine . On flat terrain, rows oriented NW-SE increased light interception and fruit overexposure in the afternoon and E-W reduced light interception and fruit overexposure in the afternoon . The high exposure to direct sunlight in NW-SE oriented rows resulted in simulated berry temperatures up to 7.8◦C higher on the SE side compared to the NW side . Compared to rows oriented NE-SW, best indoor plant pots rows oriented NW-SE had an additional 3 hours of canopy light interception above 200 W m−2 between 14:00 and 17:00 and berry temperatures greater than 35◦C for 2 additional hours . Narrow spacing affected berry temperature by potentially reducing the duration of berry exposure due to shading from neighboring vines.

Compared to the wider row spacing, the berries in narrow row spacing in N-S rows on a flat terrain intercepted up to 36% less sunlight and reduced elevated berry temperatures on the west-facing side . In the E-W row orientation, the number of hours with berry temperatures greater than 35◦C was also reduced with the narrow row spacing due to the shading from neighboring vines . The most balanced sunlight exposure and growing degree hours between each side of the vine was achieved in the N-S row orientation, although notable hourly berry temperature differences were present for both narrow and wider row spacing. For example, west-facing berries exceeded35◦C for about 1 hour for the narrower row spacing and about 3.6 hours for the wider row spacing .Adding a 30◦ slope to each of the simulations had a considerable effect on berry temperature, and could significantly change its behavior relative to flat terrain. Furthermore, the conditional inference tree results showed that the slope aspect had the strongest effect on the imbalance in temperature between different sides of the vines . In general, sloping to the south or west tended to increase light interception, berry temperatures, and berry temperature imbalance relative to north- or east-facing slopes. For example, compared to the vines oriented NW-SE on flat terrain, the vines oriented NW-SE on a southwest-facing slope increased the number of hours of Tberry > 35◦C up to 1.25 h on the southwest side . In contrast, situating these vines on a northeast-facing slope decreased the number of hours of Tberry > 35◦C up to 2 h on the northeast side . 、

For N-S oriented vines, slope had a minimal effect on daily integrated quantities such as daily light interception and berry growing degree hours, but did significantly affect short-term increases in berry temperature. N-S rows on west-facing slope increased the number of hours of Tberry > 35◦C for west-facing berries by over 2 h relative to flat terrain. However, these effects tended to be averaged out in N-S rows to maintain good symmetry over a daily period. The NE-SW oriented vines maintained good protection from berry temperature spikes and relatively good symmetry between sides of the vine as slope was added. For NE-SW oriented rows, the northwest-facing slope had more hours with Tberry > 35◦C than for the southeast-facing slope, but the opposite was true for daily-integrated quantities. This illustrates that exposure due to partially western-facing slope was more effective at generating temperature extremes than a partially southern-facing slope, but the opposite is true for daily-integrated quantities.The efficacy of shade cloth in reducing or equalizing berry temperature strongly depended on the row orientation and slope aspect . In general, adding shade cloth to the side of the row with partial or full south or west exposure tended to produce a significant reduction in berry temperatures and heat accumulation, as is to be intuitively expected. Adding shade cloth to sides of the vine with partial or full north or east exposure typically had weaker effect, and could actually increase temperatures on north-facing berries due to trapping of energy transmitted from the south. While avoiding fruit overexposure reduces fruit temperature, in some cases, controlling the amount of direct radiation received by berries with shade cloths consistently maintained the berry temperature below 35 ◦C.

For instance, in vines oriented N-S and NW-SE with wider rows, 50%and 70% shade cloth significantly reduced the time berry temperature was above 35◦C late in the afternoon in west facing berries . It was possible in several cases to achieve near-equal heat accumulation between sides of the vine while also minimizing berry temperature extremes by applying shade cloth to one side of the vine. For example, applying 70% shade cloth to the SE side of the vine in NE-SW oriented rows on flat terrain effectively balanced heat accumulation while also eliminating berry temperatures above 35◦C. E-W oriented rows always had high imbalance in heat accumulation regardless of shade cloth density or slope aspect.Comparisons between measured and modeled berry temperature indicated that the model is able to reproduce general spatial and temporal patterns of temperature, and can capture the additional effects of shade cloth. This is in addition to prior validation efforts demonstrating excellent model performance in the absence of shade cloth. Experimental validation of 3D, spatially explicit models is complicated by high sensitivity of localized model predictions to specifics of the canopy geometry. However, overall close agreement between measurements and model predictions in an average sense suggested that the model is robust to variation in vineyard architecture, topography, and the addition of shade cloth. For model validation purposes, local measurements of ambient berry microclimate were used to drive simulations. Effects of large-scale microclimatic variation was not included within this model, which could affect the predictive ability of the model as large-scale features are varied such as topography. Variation in topography could induce changes in wind speed or sensible heating of the air independent of vineyard structure, which was not represented in the model. However, radiation exposure is the primary driver of berry temperature deviations from ambient, and other microclimatic effects due to large-scale topography are likely to be secondary and establish the baseline temperature state similar to changing weather.The results of this study for flat terrain largely confirmed conclusions of previous work regarding design of vertically-trained vineyards for berry temperature management, but revealed some additional trade-offs for consideration. Similar to previous findings, the NE-SW row orientation on flat terrain is likely to be the best compromise between canopy and berry light interception, reduction of elevated berry temperatures, and balancing of heating between opposing sides of the vine, which was also argued by Tarara et al.. A trade-off of this vineyard design is that it modestly reduces overall vine light interception relative to the more common N-S row orientation. Additionally, there are still significant differences in berry heat accumulation and exposure between sides of the vine in a NE-SW row orientation. However, for VSP vineyards on flat terrain with no shade cloth, the NE-SW row orientation appeared to be the best overall at equalizing exposure between sides of the vine and reducing berry temperature extremes. For N-S oriented rows on flat terrain, previously well-documented imbalances in berry temperature between sides of the vine were also observed. It is intuitive to understand that the higher air temperatures and lower humidity that occur in the afternoon, blueberry container size when combined with berry exposure to the west sun, creates higher berry temperature than in the morning when ambient conditions are cooler. There is strong evidence that the accumulation of berry anthocyanin is a function of temperature and light Buttrose et al., Downey et al., Hunteret al., Spayd et al. and that the temperature difference between sides of the vine can create imbalance in the mass of the berries, as well as on tritable acidity, pH and phenolic compounds.

If row access by mechanical equipment is not a concern, decreasing row spacing could offer some protection against berry temperature extremes, although this is not effective at balancing opposing sides of the vine. Interestingly, results indicated that although there was high berry temperature imbalance localized to the afternoon, daily integrated metrics such as daily growing degree hours and daily berry light interception were almost perfectly balanced between sides of the vine in N-S rows. However, it is possible this was coincidental, or that abnormal diurnal temperature fluctuations such as that caused by clouds could break this symmetry. The NW-SE row orientation on flat terrain resulted in the most elevated berry temperatures. Berries on the southwest side of the vine spent nearly 4 hours above 35◦C, and shade cloth did little to mitigate these temperatures because the sun was nearly perpendicular to the shade cloth at the hottest time of day. Most previous work examining the effects of shade cloth does so for a single site and vineyard design, but results indicated that details of topography and vineyard architecture can have a significant effect on shade cloth performance. In N-S oriented rows on flat terrain, smaller row spacing relative to canopy height significantly reduced the hours of berry exposure to direct sunlight in the east and west side of the vine due to shading from neighboring vines. While berry temperatures were reduced in vineyards with narrower row spacing, grape and wine quality could decline at some point when row spacing is reduced due to excessive berry shading. Mechanical equipment access may be impeded below some threshold row spacing. Full-size equipment generally requires a minimum row spacing of around 3 m for single canopy systems. Thus, depending on the availability of equipment for mechanization and the vineyard design, shade cloth appeared to be a viable option for mitigation of berry overexposure in widely spaced rows. This study considered only VSP trellis systems at a single fruiting height, which resulted in the potential for high fruit exposure. Other trellis systems that reduce berry exposure are becoming more popular in warm climate regions. However, since it is usually undesirable to completely shade clusters because of its negative effect on berry quality, it is still necessary to understand the interaction effects between canopy architecture and berry exposure. While the results of this work can provide some initial guidance in this regard, future work analyzing different trellis types is still needed. Because of the spatially explicit nature of the model presented in this work, it is likely that only minimal adjustments to the model are needed to accommodate different trellis types.For most cases, it was observed that planting on a slope fully or partially facing south or west increased berry exposure and elevated temperatures relative to north- or east-facing slopes or flat terrain . Furthermore, a west-facing slope tended to increase temperatures more relative to a south-facing slope. This is intuitive given that the sun spends most of the day to the south, and the sun is to the west during the warmest time of a typical day. In several cases, slope had the negative effect of increasing the imbalance in heat accumulation between sides of the vine. This was especially true for the E-W row orientation, which caused very large imbalance that could not be effectively mitigated by shade cloth. For N-S and NE-SW oriented rows, the impact of slope on the berry temperature metrics was generally small. Shade cloth was able to mitigate the negative effects of slope in many cases. Applying 70% shade cloth in the sloped cases achieved excellent balance in heat accumulation between sides of the vine with N-S, NW-SE, and NE-SW orientations. The 70% shade cloth was also able to reduce the time above 35◦C to 1 hour or lower in all but the case with N-S rows on a west-facing slope, and NW-SE rows on flat terrain. The 3D model developed in this work was able to represent the effects of shade cloth on berry temperature and, thus, provided a viable tool for quantification of interactions between hypothetical vineyard designs and shade cloth on metrics related to berry temperature.

The presence of topologically non-trivial electronic band structures in condensed matter systems leads to a number of unusual consequences. A rich variety of phenomena have been discovered in gapless topological materials, such as those exhibiting Dirac-fermion excitations near the points of linear crossings of bands close to the Fermi energy EF. The breaking of either spatial inversion symmetry or time reversal symmetry splits the degeneracy of the Dirac points, leading to a pair of topologically protected Weyl points. Weyl fermions have been found to cause distinct experimental signatures, such as the chiral anomaly in transport measurements, a topological Hall effect, and Fermi arcs. Weyl fermions have mainly been studied in weakly correlated electron systems, while strong electronic correlations are frequently found to lead to novel electronic properties beyond those of simple metals or insulators, and heavy fermion systems are the prototype examples showing phenomena characteristic for strongly correlated electron systems. Here, due to strong Kondo coupling between the f-electron and conduction-band states, below the Kondo temperature , the electronic bands in the vicinity of EF may become strongly renormalized, showing a strong f-character and a huge enhancement of the quasiparticle mass. When the chemical potential lies within the hybridization gap, insulating behavior is found at low temperatures and in the topological Kondo insulators, such as has been proposed for SmB6, square plant pots the resulting electronic structure is topologically non-trivial, again leading to conducting states on the surface.

It is therefore of particular interest to look for topological heavy fermion semimetals with gapless excitations, i.e. Weyl fermions in the presence of strongly renormalized bands. Such a Weyl–Kondo semimetal phase has been predicted from calculations based on the periodic Anderson model with broken inversion symmetry. While it was proposed that Ce3Bi4Pd3 displays the low-temperature thermodynamic signatures of a Weyl–Kondo semimetal, other signatures of Weyl fermions such as the chiral anomaly have not been reported. A Weyl heavy fermion state was also proposed for CeRu4Sn6 from ab initio calculations, but no experimental evidence for Weyl fermions has been demonstrated. Consequently, whether Weyl fermions exist in the presence of a strong Kondo effect needs to be determined experimentally. Furthermore, the influence of electronic correlations on Weyl fermions is to be explored, specifically how such a system evolves from high temperatures, where the f-electrons are well localized, to low temperatures where there is a strong Kondo interaction and a reconstruction of the electronic bands. It was recently found that the half-Heusler GdPtBi, which has a strongly localized 4f-electron shell, shows evidence for Weyl fermions in an applied magnetic field due to the presence of the chiral anomaly and topological Hall effect. Here, we examine the isostructural compound YbPtBi. Although at high temperatures the Yb 4f-electrons are localized similar to GdPtBi, upon cooling YbPtBi becomes a prototypical heavy-fermion semimetal, where the enormous Sommerfeld coefficient of γ ≈ 8 J mol−1 K−2 demonstrates the enhanced effective mass of the charge carriers. This compound is therefore highly suited to look for Weyl fermions, which are strongly affected by electronic correlations. In this work, we report evidence for Weyl fermions in YbPtBi, where the bands hosting the Weyl points are strongly modified as the Kondo coupling strengthens at low temperatures.

Electronic structure calculations and angle-resolved photoemission spectroscopy measurements indicate the presence of triply degenerate fermion points in the high-temperature regime, which will each split into a Weyl node and a trivial crossing in applied fields. At these temperatures, evidence for the chiral anomaly is revealed by field-angle-dependent magnetotransport measurements. As the temperature is lowered, the chiral anomaly is not detected in the magnetotransport, but experimental signatures of Weyl fermions are found in measurements of the specific heat. This is consistent with a greatly reduced Fermi velocity due to the influence of the Kondo effect on the electronic bands near the Weyl points. Furthermore, the observation of a topological Hall effect contribution, which can arise from the Berry curvature generated by the Weyl nodes, provides additional evidence for the existence of Weyl fermions at both low and elevated temperatures.At higher temperatures, the band structure of YbPtBi can be calculated treating f-electrons as core states, as displayed in Fig. 1. The Λ6 bands cross the two hole bands near EF, forming two triply degenerate fermion points. Under amagnetic field, each triply degenerate point will further split into a Weyl point and a trivial crossing, with energies close to the bottom of the electron bands. The calculated bulk band structure with triply degenerate points is in good agreement with the ARPES results in Fig. 1b, which shows the energy–momentum dispersion relations along the surface ΓMdirection. Note that the sample can only be cleaved well with the orientation.

Along this orientation, the symmetry-equivalent bulk ΓL direction projects on the surface ΓM direction at a slanted angle, allowing for the dispersion in the vicinity of the triply degenerate points to be revealed via a careful comparison with the projected bulk band structure calculations . Two hole bands crossing EF can be clearly identified in the ARPES experiments, as well as an additional electron band with a band bottom right below EF. These experimentally observed bands are confirmed to be three dimensional bulk bands based on their photon energy dependence, and they correspond well to the theoretical calculations. The direct observation of both electron and hole pockets and their close proximity with different group velocities confirms the existence of the triply degenerate fermion points near EF, which is not affected by the slight discrepancy between the experimental results and calculations. This discrepancy is mainly related to the details of the separation and slope of the two hole bands, which could be caused by the limitations of frozen f-shell calculations and correlation effects not taken into account by the local density approximation. The good correspondence between ARPES measurements and density functional theory calculations therefore provides evidence for Weyl fermions at elevated temperatures.Based on the above experimental findings, we propose the diagram shown in Fig. 5 to describe the Weyl fermions in YbPtBi. At high temperatures there are Weyl nodes formed from the conduction bands, while the f electrons are well localized. This is consistently shown from electronic structure calculations, ARPES, and magnetotransport measurements. At lower temperatures, the strong band renormalization due to Kondo coupling enhances the effective quasiparticle mass, which modifies the dispersion ofthe bands in the vicinity of the topologically protected Weyl points, as shown schematically in the diagram. The renormalization also leads to a greatly reduced effective Fermi velocity v* compared to the bare band value, which eventually causes the disappearance of the chiral anomaly in transport measurements, but allows for the observation of a sizeable specific heat contribution C ~ 3. Importantly, there is evidence for the Berry curvature associated with the Weyl nodes from the anomalous Hall effect, which can be detected in both the intermediate and low-temperature regimes. Our results highlight the existence of Weyl fermions in YbPtBi, where we find evidence for their modification as the Kondo coupling is strengthened upon lowering the temperature. How precisely the Weyl points are modified as the electronic correlations become stronger needs to be determined by future studies. While the topological Hall effect and specific heat provide evidence for the survival of Weyl fermions at low temperatures, looking for spectroscopic evidence from ARPES or scanning tunneling spectroscopy is very important. One possible approach to reveal Weyl fermions in the heavy fermion state from f-bands is resonant photo emission. However, plastic potting pots our measurements across the Yb N edge do not show obvious resonance contrast . Although ARPES measurements with hν > 100 eV indeed reveal the bulk f bands near EF , resolving the hybridized bands deep inside the heavy fermion state is still challenging, and therefore further ARPES measurements with greater energy and momentum resolution are highly desirable. The presence of Weyl fermions in YbPtBi is different from the cases of both CeSb and GdPtBi, where the bands hosting Weyl fermions do not have a significant f-electron contribution.

Meanwhile, evidence for Weyl fermions has also been found in some magnetic d-electron systems such as Mn3Sn and YbMnBi2, where in the case of Mn3Sn a significant topological Hall effect is also observed. On the other hand, it is of great interest to look for the kind of dichotomy observed here for YbPtBi in other potential Weyl heavy-fermion semimetals, such as Ce3Bi4Pd3 where a similarly small v* was inferred from the specific heat, yet evidence for the chiral anomaly at elevated temperatures has not yet been reported. Furthermore, the strength of the Kondo interaction in heavy fermion systems can be readily tuned by non-thermal control parameters, such as pressure and magnetic field, and in particular, a quantum critical point can be reached in YbPtBi at a critical field of 0.4 T. Therefore, our findings may open up the opportunity to explore the exciting relationship between Weyl fermions, electron–electron correlations and quantum criticality.Increasing temperatures and temperature variability associated with a changing climate have become a major concern for many wine grape growing regions due to their effect on grape and wine composition. As is true for other crops, adequate sun exposure is vital because grapevines need sunlight for photosynthesis, growth, and development, and absorbed radiation by the berries is crucial for the biochemical and physiological processes that determine grape berry quality. Regrettably, excess sunlight and elevated temperatures are negatively affecting grape productivity in many growing regions. In California, minimum and maximum annual temperatures have increased from 1985 to 2011 by 2.34◦C and 1.77◦C, respectively, and in the summers by 3.88◦C and 3.31◦C, respectively . In Oakville, CA, Mart´ınez-Luscher et al. ¨ reported that elevated temperatures for grape clusters resulted in unbalanced wines with higher pH and lower levels of anthocyanins. Other research in Murrumbidgee, Australia reported that temperatures exceeding 40◦C result in delaying ripening and causing berry sunburn. Thus, strategies to minimize harmful berry temperatures are needed to sustain production in warm climates. It is challenging to manage the grapevine canopy to reduce the effect of excess temperature because of the complex interactions between plant architecture and the environment. Traditionally, it has been recommended to time canopy management to maintain sun-exposure for young fruits, but also to allow some shading of mature grape clusters to prevent excess sun exposure. Although increasing the shade in vineyards palliates the effect of elevated temperature, trade-offs need to be evaluated as well. For instance, shaded clusters can cause delay in fruit ripening, reduce wine quality, and increase disease prevalence due to pathogens such as bunch rot and powdery mildew. The negative effects of elevated temperature on grape berries could likely be mitigated in many cases if the complex interactions between canopy architecture and microclimate were better understood and predicted at the berry level. Developing and evaluating proposed mitigation strategies experimentally based on field trials is costly and time-consuming, which can limit their breadth and generalizability. Crop models can have the potential to extrapolate the results of a limited set of experiments through systematic variation of relevant variables, however, there are currently no models available that can represent varying grapevine architectures and their effect on spatial and temporal fluctuations in grape berry temperature. Through three different studies, this dissertation aims to 1) investigate the impact of the heterogeneous and anisotropic vegetation structure characteristic of grapevines on light interception, 2) develop a 3D model to simulate grape berry temperature in response to varying vineyard architecture and topography, and 3) identify strategies that have the potential to mitigate unfavorable temperatures in grape berries. Chapter 2 evaluates widely used assumptions when modeling solar radiation interception in plant canopies. The solar radiation intercepted by plant canopies is a fundamental driver of biophysical processes on Earth, and thus, quantifying such interception is a critical part of understanding and predicting a wide range of processes occurring at the land-atmosphere interface. The study showed that using a 1D model to simulate light interception for discontinuous canopies resulted in overestimation of light interception by up to 115% for the cases considered.

Previous research using mitochondrial and microsatellite DNA markers found some evidence of population structure, as well as evidence that the European invasion originated from a single population in central Chile . However, determination of higher-resolution population structure, migration events, divergence times, and population size can benefit from using a larger number of markers, such as what is produced from genome-wide sequencing studies . Additionally, few genetic studies have been conducted to understand how Tuta absoluta has performed so successfully as an agricultural pest beyond targeted examinations of known insecticide resistance alleles. One reason for this has been the lack of a highly contiguous genome with annotated genes. A short-read based assembly has been previously published for the purpose of developing molecular diagnostics ; however, it is highly fragmented and duplicated. In this study, we addressed these issues by using long-read sequencing technology to produce a highly contiguous genome assembly for Tuta absoluta. We then use short-read technology to sequence genomes of individuals collected across Latin America, as well as a Spanish population, blueberry pot to identify single nucleotide polymorphisms in an unbiased manner. We use these SNPs to detect population structure and estimate population history parameters to understand how and when Tuta absoluta spread across Latin America. Finally, we use genome scanning statistics to identify genes putatively under selection that may explain Tuta absoluta’s success as an agricultural pest. We expect the genome assembly and population data will be an asset toward developing new strategies to manage this pest.

For genome assembly, a single Tuta absoluta larva was collected from a colony originally sourced from the Institute of Agrifood Research and Technology , Cabrils, Spain and held in the Contained Research Facility in UC Davis and frozen on dry ice. The larva was pulverized in liquid nitrogen with a pestle in a 2 mL microcentrifuge tube using 740 mL of lysis buffer , 135 ug/mL Prot K. After a 37℃ overnight incubation step, 240 μL of 5M NaCl was added and gently mixed in by rocking before centrifuging at 10,000 RCF, 4℃, for 15 minutes. Supernatant was transferred using a wide-bore pipetteto a 2 mL DNA low-bind tube , precipitated with 1 mL of 100% ethanol, and centrifuged at 10,000 RCF, 4℃, for 5 minutes. The DNA pellet was washed with 500 μL of ice-cold 70% ethanol twice before air-drying for 5 minutes. Dry pellet was resuspended in DEPC-treated water and allowed to dissolve for 1 hour at room temperature before being stored at 4℃ for no more than 2 weeks. Absorbance ratios were measured with a Nanodrop Lite , DNA concentration was measured with a Qubit 4 Fluorometer using a dsDNA High-Sensitivity Assay , and DNA fragment size was measured with a Tapestation genomic DNA ScreenTape . Approximately 700 ng of DNA was sent to QB3-Berkeley for library preparation and PacBio HiFi sequencing with 1 SMRTcell.We analyzed whole-genome sequencing data from individuals previously collected from field and greenhouse sites across South America and Costa Rica, as well as a lab colony from Spain . Mapping rates to the new genome assembly ranged between 70%-90%, although sequencing depth per individual was low . One population from Argentina had extremely low mapping rates and read depth, so we excluded it from further analysis. Wherever possible, we used methods based on genotype likelihoods, rather than genotype calls, to account for uncertainty that results from the low read depth.

To investigate population structure in our samples, we used Principal Component Analysis and admixture estimation based on allele frequencies from over 900,000 SNPs. The first two PCs captured 18.7% and 16.3% of the total variance in the data, with the remaining PCs each capturing less than 5% of the total data variance . Samples primarily cluster together based on collection site but also formed three distinct regional groups . Samples from Chile, Peru, and Ecuador form an “Andes” cluster west of the Andes Mountains; samples from Brazil, Uruguay, Paraguay, and Argentina form a “Central” cluster, east of the Andes Mountains; and samples from Columbia and Costa Rica form a “North” cluster. Spanish samples grouped tightly with the Andes cluster, particularly the VA site. When three clusters were allowed in admixture estimation, samples group into the same three clusters as in PCA, while at four clusters, the Spanish samples become their own group, with VA samples sharing a large proportion of admixture. Compared to other Andes populations, the VA samples are more differentiated from Central and North sites as well, with little signal of admixture at all levels of k tested. The other Andes populations all had low admixture proportions from Central at k=2, although at k=3 we see that all RI samples exhibited admixture from the North populations. This suggests that the non-VA Andes populations are more closely related to Central populations than VA, and that VA could represent an admixture between the population that gave rise to the Spanish lineage and the other Andes populations. Additionally, we see that RI represents an intermediate population between the Andes and North, which makes sense given its geographic location between the two clusters. To further quantify population structure between these clusters, we calculated nucleotide diversity, Tajima’s D, and Fst using genotype likelihoods . For all clusters, nucleotide diversity was approximately 2%, which is fairly high compared to most Lepidopterans .

If we look at the weighted Fst, we see differentiation between clusters is high, particularly between North and all other clusters. The combination of high diversity levels and high Fst could mean these regions diverged from each other a long time ago, prior to the detection of Tuta absoluta by growers across Latin America in the 1960s to 1980s. If divergence had occurred recently, we might expect reduced diversity levels in invasive populations relative to the ancestral population.To detect potential migration events between populations, we used Treemix to build a maximum likelihood tree based on allele frequencies, as well as predict migration edges and calculate F3 statistics. As Treemix was designed to take allele count data per population, we called genotypes using PCAngsd using a 95% accuracy cutoff and counted alleles within each sampling location. After filtering out loci with missing data, 47,535 SNPs were available for use. In general, the tree topography aligns with results from PCA and admixture analyses. We see sampled sites cluster into the same three clusters, North, Andes, and Central, with the Spanish samples sister to the VA site . In agreement with Fst estimates, North populations have experienced more genetic drift from the Andes and Central populations, compared to the Andes and Central populations with each other. Interestingly, the RI site does not form a clade with other Central populations but descends from the common ancestor of the Central/Andes group. Based on admixture analysis that showed low levels of admixture in RI from the North, the position of RI in the tree could be further evidence that Ecuador represents an intermediate mixing zone between populations north and south of it. To investigate further, we re-ran Treemix allowing between one to five migration events and calculated the F3 statistic between all combinations of three populations to see if admixture was supported. At m=2,4 and 5, Treemix reported a strong migration from the Spain or Spain/VA branch to RI, while at m=3 and 5, Treemix reported a weak migration event from the North to RI. F3 statistics F3 and F3 were significantly negative , indicating that a simple bifurcating tree does not explain RI’s relationship with CH, CR, and SP. While Treemix infers a migration from the Spanish branch to the RI branch, it is important to remember that this migration is inferred to have occurred somewhere along the branch between the current day Spanish population and the most recent common ancestor of Spain and VA . This migration could have occurred early in the branch, nursery pots when the population was still in Chile, or late in the branch, when the population moved to Spain. Based on fresh tomato trade between the two countries, in 2006 Chile shipped over 29,000 kg of fresh tomatoes to Spain while importing none back . This makes admixture from Spain back to RI unlikely and suggests that RI contains admixture from North and Chilean populations. In addition to admixture in RI, Treemix and F3 statistics also detected admixture in AR from the Spanish population. At m=1,2,3, and 4, Treemix detected a migration edge from the Spanish branch to AR with migration weight varying between 9% to 17%. F3 statistics of AR and Spain with any population from North or Central resulted in a significantly negative value, providing strong evidence of a migration event from a Spanish ancestor like the signal detected with RI.

This suggests that the admixture signal we see in AR is from the same Chilean population that gave rise to the Spanish invasion and RI admixture.Based on the high levels of nucleotide diversity and Fst between the Andes, Central, and North clusters, we hypothesized that the three regions may have diverged many generations ago, before the appearance and detection of Tuta absoluta in agricultural crops throughout South America in the mid- 20th century. This would suggest a model in which Tuta absoluta may have adapted from local, wild host plants to nearby tomato fields independently, rather than a single population that became adapted to tomatoes and was spread through human activity. To investigate this, we calculated the folded two dimension site frequency spectrum between populations and estimated parameter values under various population models using maximum likelihood coalescent methods . We excluded the VA samples from the Andes cluster to avoid potential modeling issues due to VA appearing to originate from a distinct ancestor than other Andes populations. The simplest model allows for two population splits with constant population sizes, while the exponential growth model adds an exponential growth rate to each population. As exponential growth may not be appropriate if divergence times are long, we also tested a model with a simple resizing event for each population at some point in time. We used a post-hoc comparison of simulated linkage disequilibrium decay rates between models to test model fit. We found that while all three models simulated decay rates within the 95% confidence interval of the Andes population data, none simulated decay rates that overlapped with Central and North decay rate estimates, although the resizing population model was closest. Under the resizing population model, divergence of the North occurred 252,383 generations ago , followed by a Central population divergence 187,034 generations ago . Reports of Tuta absoluta generation times can be as high as 6 to 12 or more generations per year , dating these divergence events to tens of thousands of years ago. This suggests that Tutaabsoluta was already present across Latin America prior to the 1960s, and as tomato agriculture surged, adapted locally to the new host plant.The Population Branch Statistic is an Fst-based statistic that uses Fst data between 3 populations to calculate the population-specific allele frequency changes. Regions of the genome with abnormally high PBS may be under strong selective forces, causing the loci allele frequencies to change faster than expected by drift. We calculated PBS across the genome for all three populations and found several peaks in contigs 2, 9, 15, and 22 that were exceptionally high and broad, particularly in the North cluster . The peak in contig 9 contained the gene paralytic , a neuronal sodium channel protein that is the active target of pyrethroid insecticides . While PBS peaks in the North population between 13.1 and 13.2Mb on contig009, we note that the allelic diversity was low in the Andes and Central clusters relative to the North . We calculated allele frequencies of known resistance-inducing mutations in each cluster , and found one mutation, an alanine to leucine substitution at position 1014, was fixed in the Central and Andes, while at 41% frequency in the North . In addition, we found low to intermediate frequencies of other resistance alleles, including M918T, T929I, V1016G, L925M, and I254T. A similar selective sweep signal was also seen in the PBS hotspot on contig 2 , with high PBS and diversity levels in the North, and a large region of low allelic diversity in the Andes and Central .

Dynamics of pp highlighted the typical inversion phenomenon of the diel curve in olive leaves from trees under deficit irrigation . In NB, DI-66 and DI-33 leaves exhibited the half-inverted state , whereas DI-0 leaves showed a total inversion of the curve . In MN trees, a clear shift from state I to II was not observed, despite the slight tendency to enter state II at 219 and 221 DOY, with no apparent differences among irrigation levels . This suggests that MN leaves can maintain high cell turgor, probably by reduced cell wall elasticity or osmotic adjustments, as found in other olive genotypes . The highest RRfruit always occurred early in the night as fruit quickly rehydrated their tissues . As expected, the most negative RRfruit rate always occurred in the warmest hours of the day. RRfruit dynamics were also affected by deficit irrigation in NB, as the diel RANGE was greater in DI-0 and DI-33 than in DI-66 and FI fruit . A completely different behavior was observed in MN fruit, which instead had the widest diel RANGE in FI trees . In addition, the overall diel RANGE of RRfruit in MN was almost double than in NB, implying larger water in- and out-flows per unit of fruit volume in the former, determined by high fruit sink power for water. A general positive peak of RRleaf was exhibited early in the morning , representing a quick leaf turgor loss after pre-dawn highest turgor in the 24-hour timeframe. Even in this case, the two cultivars responded differently to water deficit, growing blueberries in pots with NB DI-0 trees exhibiting minimal diel fluctuations while MN DI-0 trees showing the largest RANGE. This suggested that the oscillations of RRleaf might be linked to those of RRfruit. Another 5-day interval was considered at stage III of fruit development .

Differently from stage II, FD responses were characterized by an evident diameter increase across the 5 days and within the 24-hour period in both cultivars, as in stage III fruit are in full cell enlargement phase . Daily curves of pp did not show pronounced inversion phenomena, as this week was characterized by high rainfall and general higher midday 9 stem . Only NB DI-0 trees showed a partially inverted pp curve. Diel RANGE of RRfruit was found to be greatly reduced at stage III compared to stage II . In the former, low VPD and good soil water availability determined by abundant precipitations led to an increase of water content in fruit and lower fruit water exchanges. For similar reasons, the diel RANGE of RRleaf was reduced in stage III , although NB and MN maintained the same differences in response to deficit irrigation levels observed at stage II . Considering the interesting findings from RRfruit and RRleaf dynamics, these two indices were further related to each other regressing their diel data at 15-minute intervals in a day at stage II and stage III . Scatter plots in Figure 10 show anti-clockwise hysteretic relationships between RRfruit and RRleaf, both for NB and MN . Hysteresis are common when relating outputs from different sensors of plant water status mounted on different organs , as there is generally a lag in tissue water de- and re-hydration, and in our case, also a likely different pattern of the RRleaf to RRfruit relationship between day and night. An overall decrease of the hysteretic loop area occurred from stage II to stage III in both cultivars . This is probably driven by the different fruit growth pattern at stages II and III which induced a reduction of the RRfruit diel range . In both DOY 223 and 287, the hysteretic loops in NB progressively flattened along the RRfruit axis with increasing water deficit due to the change in the ratio between RRfruit and RRleaf.

In other words, on one hand, at increasing water deficit and in a diel interval, it seems that NB leaves significantly reduce water exchanges, as the values of RRleaf stay around 0 Pa kPa−1 min−1 . On the other hand, increasing water deficit caused MN loops to flatten along the RRleaf axis , with MN leaves keeping high water exchanges at low 9 stem, while fruit water exchanges were significantly reduced, as RRfruit did not change much from 0 µm mm−1 min−1 .This opposite trend suggests a completely different mechanism of leaf and fruit water exchanges in response to increasing water deficit in the two cultivars, which might be driven by different osmotic adjustments, cell-wall elasticity and tissue water content. The statistical diel, nocturnal and diurnal parameters of RRfruit were associated to the corresponding RRleaf parameters to assess fruit and leaf responses to water deficit. Subsequently, data were analyzed by MANOVA to determine whether the combined response of parameters was affected by cultivars, irrigation levels, and cultivar × irrigation interaction. The cultivar did not influence significantly diel, diurnal and nocturnal RRfruit/RRleaf when statistical parameters were considered together . Diel and diurnal RRfruit/RRleaf parameters changed significantly in response to irrigation levels, but the cultivar × irrigation interaction had the strongest effect , indicating that RRfruit/RRleaf responses to deficit irrigation differ between the two genotypes under study. Specifically, the highest F was found in the MANOVA that tested diurnal RRfruit/RRleaf responses to cultivar × irrigation. These results suggest that, under increasing water deficit, the differences in genotype-specific fruit and leaf sink power to water are predominant in day hours.Human persistence depends on many natural processes, termed ecosystem services, which are usually not accounted for in market valuations. Global degradation of such services can undermine the ability of agriculture to meet the demands of the growing, increasingly affluent, human population . Pollination of crop flowers provided by wild insects is one such vulnerable ecosystem service , as their abundance and diversity are declining in many agricultural landscapes . Globally, yields of insect-pollinated crops are often managed for greater pollination through the addition of honey bees as an agricultural input . Therefore, the potential impact of declines in wild pollinators on crop yields is largely unknown, as is whether increasing application of honey bees compensates for losses of wild pollinators, or even promotes these losses. Wild insects may increase the proportion of flowers that develop into mature fruits or seeds , and therefore crop yield , by contributing to pollinator abundance, species number , and equity in relative species abundance . Increased pollinator abundance, and therefore visitation rate to crop flowers, should augment fruit set at a decelerating rate until additional individuals do not further increase , or even decrease fruit set . Richness of pollinator species should increase the mean, and reduce the variance, of fruit set , because of complementary pollination among species , facilitation , or “sampling effects” , among other mechanisms . Pollinator evenness may enhance fruit set via complementarity, or diminish it if a dominant species is the most effective pollinator . To date, drainage gutter the few studies on the importance of pollinator richness for crop pollination have revealed mixed results , the effects of evenness on pollination services remain largely unknown, and the impact of wild insect loss on fruit set has not been evaluated globally for animal pollinated crops.

We tested four predictions arising from the assumption that wild insects effectively pollinate a broad range of crops, and that their role can be replaced by increasing the abundance of honey bees in agricultural fields: for most crops, wild-insect and honey bee visitation enhances pollen deposition on stigmas of flowers; consequently, for most crops, wild insect and honey bee visitation improves fruit set; visitation by wild insects promotes fruit set only when honey bees visit infrequently ; and pollinator assemblages with more species benefit fruit set only when honey bees visit infrequently . To test these predictions we collected data at 600 fields on all continents, except Antarctica, for 41 crop systems . Crops included a wide array of animal-pollinated, annual and perennial fruit, seed, nut, and stimulant crops; predominately wind-pollinated crops were not considered . Sampled fields were subject to a diversity of agricultural practices, ranging from extensive monocultures to small and diversified systems , fields stocked with low to high densities of honey bees , and fields with low to high abundance and diversity of wild insects . For each field, we measured flower visitation per unit of time for each insect species, from which we estimated species richness and evenness . We quantified pollen deposition for 14 systems as the number of pollen grains per stigma, and fruit set for 32 systems as the percentage of flowers setting mature fruits or seeds. Spatial or temporal variation of pollen deposition and fruit set were measured as the coefficient of variation over sample points or days within each field . The multilevel data provided by fields within systems were analyzed with general linear mixed-effects models that included crop system as a random effect, and wild-insect visitation, honey bee visitation, evenness, richness, and all their interactions as fixed effects. Best-fitting models were selected based on Akaike’s Information Criterion . In agreement with the first prediction, crops in fields with more flower visits received more pollen on stigmas, with an overall 74% stronger influence of visitation by honey bees than by wild insects . Honey bee visitation significantly increased pollen deposition in seven of ten crop systems, and wild insects in ten of 13 systems . Correspondingly, increased wild insect and honey bee visitation reduced variation in pollen deposition among samples . Contrary to the second prediction, fruit set increased significantly with wild-insect visitation in all crop systems, but with honey bee visitation in only 14% of systems . In addition, fruit set increased twice as strongly with visitation by wild insects than by honey bees . These partial regression coefficients did not differ simply because of unequal abundance, or disparate variation in visitation between wild insects and honey bees. In crop systems visited by both honey bees and wild insects, honey bees accounted for half of the visits to crop flowers , and among-field CVs for visitation by honey bees and by wild insects were equivalent. Furthermore, wild-insect visitation had stronger effects than honey bee visitation, regardless of whether honey bees were managed or feral and, comparing across systems, even where only wild insects or honey bees occurred . Moreover, wild-insect visitation alone predicted fruit set better than honey bee visitation alone . Correspondingly, the CV of fruit set decreased with wild-insect visitation, but varied independently of honey bee visitation . This contrast likely arose from pollen excess, filtering of pollen tubes by post pollination processes, and seed abortion , and so reflects pollination quality, in part. Intriguingly, the difference in coefficients between pollen deposition and fruit set for honey bees greatly exceeds that for wild insects , indicating that wild insects provide better quality pollination, such as greater cross-pollination . These results occurred regardless of which crop systems were selected , sample size , the relative frequency of honey bees in the pollinator assemblage among systems, the pollinator dependence of crops, or whether the crop species were herbaceous or woody, or native or exotic . Poor-quality pollination could arise if insect foraging behavior, based on focal resources typical of honey bees , causes pollen transfer between flowers of the same plant individual or the same cultivar within a field, thereby limiting cross pollination and increasing the incidence of self-pollen interference and inbreeding depression . The smaller difference in coefficients between pollen deposition and fruit set for wild insects, and the stronger effect on fruit set of wild-insect visitation, suggest that management to promote diverse wild insects has great potential to improve global yield of animal-pollinated crops. The third prediction was also not supported, as fruit set increased consistently with visitation by wild insects, even where honey bees visited frequently . In particular, the best-fitting model for fruit set included additive effects of both visitation by wild insects and honey bees , suggesting that managed honey bees supplement the pollination service of wild insects, but cannot replace it. Overall, visitation by wild insects and honey bees were not correlated among fields , providing no evidence for either competition for the resources obtained from crop flowers , or density compensation between wild insects and honey bees at the field scale.

However, we also observed upregulation of biosynthetic genes involved in production of trichothecenes , which indicates that F. acuminatum also relies on other toxins during infection of tomato fruit concordant with the classification of F. acuminatum as strong toxin producer . Additionally, the AA6 family that appears during RR infections of F. acuminatum and R. stolonifer may be involved in metabolism of host defense compounds. These enzymes are 1,4-benzoquinone reductases, which have been shown to function in fungal protection against destructive host-produced quinones . Another physiological factor which may influence the success of infection is the pH of the pathogen-host interface. As the tomato fruit ripens, the apoplast becomes more acidic . Furthermore, B. cinerea has been shown to acidify the host environment through the production and secretion of oxalic acid . A key enzyme in oxalic acid biosynthesis is BcOAH1 , which encodes oxaloacetate hydrolase . This gene is not upregulated during interaction with tomato fruit in any of the treatments. However, there is significant downregulation of this gene in RR fruit compared to MG fruit. This suggeststhat, if B. cinerea utilizes oxalic acid to acidify tomato fruit, it does so to a much lesser extent in RR fruit where the pH is already comparatively acidic. In contrast, during infection of Arabidopsis roots, F. oxysporum relies on alkalinization via peptides known as rapid alkalinizing factors . However, a BLAST search of RALF sequences, as was performed to identify fungal RALFs in Thynne et al. , square pot revealed no clear RALF genes in our transcriptome of F. acuminatum. The importance of fruit ripening for the success of fungal infections was confirmed by comparing fungal growth and disease development in fruit from wild-type and a non-ripening mutant after fungal inoculation.

Growth and morphology of B. cinerea, F. acuminatum and R. stolonifer on nor MG and RR-like tomato fruit was comparable to that on wild-type MG fruit. This result is in agreement with our previous report that nor tomato fruit is resistant to B. cinerea infections . The inability to infect non-ripening tomato fruit highlights the dependency of these fungi on the activation and progression of ripening events that transform the host tissues into a favorable environment for disease development. Altogether, our results confirm that infection success of the three pathogens B. cinerea, F. acuminatum and R. stolonifer largely depends on fruit ripening stage. This is due to all three pathogens sharing similar lifestyles and necrotrophic infection strategies. However, the capacity to infect different plant tissues differs between the three fungi. B. cinerea shows distinct strategies in both ripening stages likely due to its ability to induce susceptibility in the host , whereas R. stolonifer is active almost exclusively in RR fruit. The ability of F. acuminatum to infect both MG and RR fruit may be reflective of its especially wide host range, which includes insects in addition to fruit . A summary of infection strategies utilized by the three pathogens during infection of MG and RR tomato fruit is shown in Table 2. Further research on which processes identified are required for successful infection would lead to a greater understanding of fruit-pathogen interactions and, ultimately, strategies for their management.The tomato is a functional genomics model for fleshy-fruited species and is one of the most popular and economically important crops globally . However, storage at temperatures below 12.5°C followed by rewarming to room temperature, compromises fruit quality, hampering the post harvest handling of this commodity .

This cold-induced damage to the fruit called post harvest chilling injury may only be detectable as a loss of flavor, or in severe cases, as fruit spoilage, the extent of which depends on the storage temperature, length of exposure, genotype and fruit developmental stage . The progression of PCI in fruit tissues is complex. It is marked by a loss of selective membrane permeability, increased solute leakage, reactive oxygen species accumulation and metabolic dysfunction . After the fruit is transferred to room temperature for rewarming or reconditioning, higher respiration ensues within days , and within a week, secondary symptoms such as uneven color formation, surface pitting,water soaking and decay are visible . Symptoms are more intense in green compared to riper fruit, since maturation processes are disrupted by chilling . Because of the negative effect on tomato quality and shelf-life, our goal is to better understand PCI development and regulation in this species. First, we investigated the spatial and temporal evolution of PCI in the whole tomato fruit using MRI. Most studies of tomato PCI have focused on the pericarp, ignoring the internal tissues, which can account for 30% and 70% of the fresh mass of round and cherry tomatoes, respectively. Tao et al. , investigated changes in chilled ‘Micro-Tom’ fruit using non-invasive MRI. They showed that the columella and locular region differed from the pericarp in their response to cold, which has implication for understanding the underlying causes of PCI. The fruit in that study were subjected to a severe cold stress , since this genotype is not as sensitive to chilling temperatures as many commercial varieties . Further, only one developmental stage was chosen . It is not known if their findings are applicable to other cultivars, storage conditions or maturation stages. Second, we investigated if 5-azacytidine could alter PCI. This chemical inhibits DNA and RNA methylation , epigenetic modifications that regulate gene expression, in response to developmental and environmental stimuli in a tissue-specific manner .

DNA methylation is a key regulatory process for tomato fruit ripening ; injecting AZA in round tomato fruit accelerated ripening . It was shown that chilling-induced reductions in red fruit volatiles correlated with methylation of key ripening genes. Co-regulation of the ripening and cold response regulatory networks in fruit undergoing chilling stress seems likely . Since differential methylation is essential to both processes, we wanted to determine if AZA could influence PCI symptoms in tomato fruit. In this study, two questions were asked: 1) is it possible to detect spatio-temporal differences in chilled tomato fruit differing in maturation stage, and temperature × time of storage by low-resolution MRI?, and 2) would AZA influence PCI response? For the former, we used commercial cherry tomatoes and mild to moderate chilling stress. For the latter, fruit were injected with AZA weekly in order to detect changes in PCI by methylation , specifically on respiratory activity. Fruit from a commercial cherry cultivar and the functional genomics model ‘Micro-Tom’ were used in this study.At this developmental stage in ‘Sweet 100’, the pericarp, columella and locular tissue showed a differentiated pattern in terms of their D-values after 7 days of chilling . Values were highest in the pericarp followed by the locular tissue and columella. Similar patterns were seen in freshly-harvested breaker fruit . These three tissues have heterogeneous transcriptional and metabolic profiles due to their distinct origin and functionality . This likely contributed to the distinct D-values observed. When D-values for each region were compared as over each chilling period, no changes were observed except for the columella in fruit held at 5°C. Unchanged D-values may be due to cold-induced reductions in free water movement within tissues, and pectin solubilization . Fruit exposed to warmer temperatures, i.e., after storage at the control temperature for 7 days, or after transfer from the cold to 20°C, showed more dynamism in D-values. The different tissue fractions, which had distinct D-values during chilling, changed and became more similar when exposed to warmer temperatures . These non-chilling temperatures may have allowed ripening and other physiological eventsto take place, leading to these changes.Figure 3A shows the D-values of ‘Sunsugar’ ripened fruit. These data, gathered from breaker, pink, square plastic planter and red fruit immediately after harvest, suggest that as ripening progresses, the D-values of the columella and locular region become more similar . Ripening increases the proportion of free water and metabolites within tissues, due to liquefaction of the locules and breakdown of the structural components of the cell . These changes may have underscored the increased Dvalues seen here, and in other studies . A similar occurrence was seen when red fruit was stored at 2.5°C for 5 days . When D-values for each region were compared over time, there was no significant difference. Tissue liquefactionin red fruit was so extended as a consequence of ripening, that cold did not generate any detectable increase by the MRI, or did not increase membrane leakage since it was already fluid. The observations of pink fruit stored in the cold and then rewarmed are less clear. Both chilling-induced damage during low-temperature storage, and ripening-related tissue deconstruction during rewarming would lead to increased membrane permeability and Dvalues , thus making it difficult to attribute higher Dvalues to one or the other biological phenomenon.

There are some points to emphasize with respect to the data when analyzed across cultivars and conditions. First, pericarp D-values did not vary as much as those in the columella and locular regions . Second, there was a weak correlation between MRIderived values for the pericarp and the physical changes caused by cold, visible on the pericarp e.g., poor color development, pitting and decay as reported by the CII data . In contrast, there was more synchrony for the columella and CII which is similar to the data published by Tao et al. . Surprisingly, the locular fraction showed a similar r-value to the pericarp when CII was considered. Therefore, other mechanisms besides the increased water mobility we were able to detect under the experimental conditions used, may have a higher contribution to the development of chilling induced external symptomatology. Third, different D-values were recorded in the three tissues as ripening progressed: they decreased in the pericarp, increased in the columella and were unchanged in the locular tissue , exemplifying the unique response of each tissue-type. Fourth, MRI could only detect changes after transfer of chilled fruit to room temperature. Loss of membrane selective permeability due to a cold-induced membrane phase transition was not sufficiently advanced to produce detectable increases in free water mobility during cold storage. This supports the view that, increased membrane permeability is unlikely to be one of the earliest events in PCI response, but occurs at a significant rate during rewarming .Fruit undergoing PCI normally exhibit a transitory burst of CO2 when transferred from chilling to room temperature, which acts as a reliable marker for the early stages of cold injured tissue . If AZA-treated fruit show differences in respiratory activity after cold stress compared to the water-treated fruit, this could be indicative of an effect of methylation on PCI. Different responses were observed across varying cold stress in ‘Micro-Tom’ and ‘Sun Cherry’ and are described in turn.MRI was useful for detecting fruit ripening, its attenuation by cold, and fruit tissue specificity in the cold response. MRI non-invasively differentiated among tomato pericarp, columella and locular fractions. However, when fruit were scanned after reconditioning, or when at an advanced stage of ripening, these distinctions were lost. Chilling-induced damage was detected by the MRI in the columella but not in the pericarp and locular tissue. MRI scans in the columella throughout the experiment better reflected the CII. Cold stress likely repressed the mechanisms leading to fruit free water production or increased water mobility. AZA was used to determine if demethylation could modulate the effect of PCI in chilled tomato fruit. The effect of AZA on PCI was determined by many multilayered factors, e.g., genotype, severity of stress and how it influences the underlying ripening pathways. This complexity is probably a consequence of the ubiquity of epigenetic methylation on the genome and transcriptome and the multitude of factors that influence its status. Even though in some conditions, the effect of AZA was not detected, it does not mean that methylation is not important to PCI, since the phenotypes assessed probably do not reflect all methylated regulation of fruit gene expression.Strawberry is an important soft fruit crop that is grown worldwide on more than 370 000 hectares and, for the United States alone, the total value of the annual strawberry production exceeds US$2.3 billion . Strawberries are beneficial to the human diet as a source of macro- and micronutrients, vitamins and health promoting antioxidants . Strawberry is a perennial herbaceous plant with short stems and densely spaced leaves. Strawberry produces complex accessory and aggregate fruit composed of achenes and a receptacle .

The evolutionary and genetic aspects of the process of domestication have been studied intensively in temperate cereal crops and pulses and are fairly well understood for those crops . In contrast, we have a much less comprehensive knowledge of domestication in perennial crops such as tree fruits and nuts , even though these species make up an important component of human nutrition throughout the world . It has been suggested that the study of tree crops may reveal fundamental differences in the process of domestication and its genetic consequences . The fact that tree crops have long lifespans and that they are often asexually propagated limits the number of generations that have passed since their first cultivation by humans and the degree of evolution that has occurred under human selection in these taxa . Clement described five stages in the domestication process: 1) wild , 2) incidentally co-evolved , 3) incipiently domesticated , 4) semi-domesticated , and 5) domesticated . In species that are still in the early stages of domestication , cultivated and wild-type individuals will coexist in close proximity in the region of origin, where we can study how traits have responded to anthropogenic selection in the face of continued gene flow between wild and improved types. Some of our best-known examples of plants in the early stages of domestication come from Mesoamerica, where native crop species are frequently cultivated in traditional agroecosystems such as home gardens . We expect fruit and seed characteristics of tree crops to change in predictable ways under anthropogenic selection during the domestication process, 25 liter pot leading to a type of domestication syndrome for edible fruits .

Compared to their wild relatives, edible fruits of domesticated taxa tend to be larger and sweeter or higher in oil content . The ratio of edible product to waste, or “economic ratio” , goes up. We also expect to see a reduction in toxic compounds that confer defense against natural enemies at the cost of palatability . In addition to changing mean trait values, anthropogenic selection could have contradictory effects on the variance of traits. Due to selection bottlenecks, we expect neutral variation to decline with cultivation in domesticated species . For particular genes under strong directional selection, the reduction in genetic variation may be even more dramatic . However, if distinct cultivated varieties are selected for contrasting phenotypes, then phenotypic variance will be higher in cultivated plants . Some even have used changes in the variance and shape of the distribution of traits as an indicator of the degree of domestication shown by populations of indigenous fruit trees . Chrysophyllum cainito L. , commonly known as caimito or star apple, is highly desired throughout the tropics for its value as anornamental tree and its production of large, edible fruits. Caimito is presently cultivated throughout the Caribbean, Central America, and parts of South America as well as in Southeast Asia . Various hypotheses have been suggested for its geographic and taxonomic origin, as well as its center of domestication . Historical accounts made by plant explorers in the 16th and 17th centuries mention C. cainito as occurring in the islands of the Lesser and Greater Antilles as well as on the mainland and islands of the Bay of Panama . Patrick Brown mentions C. cainito as being cultivated all over the island of Jamaica and that it “thrives with very little care” . Seemann reports the presence of both wild and cultivated caimito in the Isthmus of Panama. Our preliminary molecular genetic results show much greater overall diversity of the species in Panama compared to northern Central America, Mexico, and the Caribbean, and support the hypothesis that C. cainito was domesticated in Panama.

In contemporary Panama, C. cainito is widely distributed as a natural part of tropical lowland forest. In fact, this species is promoted as a native tree for environmentally sensitive reforestation efforts, for example, by the Proyecto de Reforestación con Especies Nativas . The forest plot network that bisects the Panamanian Isthmus documents the presence of C. cainito at many sites on the Pacific side of central Panama . In the wild, C. cainito is found at low density, does not produce fruit until the tree reaches the canopy of the forest, maintains few branches below 10 m, and produces ripe fruits over only a short time interval; taken together, these aspects complicate the study of this species in wild populations. Cultivated caimito is a common component of Panamanian home gardens and small ranchos. Usually only one or two individuals are planted per property, as fruits are not sold on a commercial scale. While an owner will occasionally report that a tree “arrived on its own,” presumably dispersed by a bird or mammal, usually trees in gardens have been planted from seeds selected from a fruit, usually a fruit provided by a friend or neighbor . Sometimes plants have been purchased as seedlings from a nursery. During the Canal Zone era , C. cainito was planted horticulturally as a street tree in some communities within and surrounding Panama City. Thus the cultivation of C. cainito trees in Panama has included a combination of individual selection via fruits and seeds, with some relatively small-scale central production and distribution of plants. In addition, human migration between the Antilles and Panama may have resulted in the importation of caimito seeds from the Antilles. Our goal was to test the hypothesis that cultivated individuals of C. cainito show signs of a domestication syndrome: Distinctive fruit and seed characteristics that are typically associated with domestication relative to wild phenotypes growing in the same region. As a putative region of domestication for this species and an area with extensive populations in protected forests, Panama is the ideal place for such a study. We compared fresh collections from wild and cultivated trees in central Panama for a range of morphological and fruit quality traits to infer how human selection has influenced these traits. We then used Discriminant Analysis to investigate the relative importance of various traits for distinguishing cultivated fruits from wild fruits. In this paper, we also present information on the variance in traits, correlations among traits, and variation in trait values from year to year.We chose to focus our collections in central Panama, where caimito is cultivated in both rural and urban communities set within the context of wild populations in protected forests. Central Panama includes extensive areas of semi-deciduous, tropical forest, primarily associated with the Panama Canal watershed. There is a natural rainfall gradient of 1,750–3,000 mm per year from the Pacific to the Caribbean side of the isthmus, with a pronounced dry season .

Wild individuals of Chrysophyllum cainito are distributed at low density throughout the forestsof the Canal Zone, particularly on the Pacific side of the continental divide, where they are found in old-growth as well as second-growth forests . For the purpose of our sample, we defined trees as wild if they were in natural forest habitat and if, to the best of our knowledge, people did not plant them. We used surveys and the knowledge of local indigenous people to locate over 150 adult wild trees, but only a percentage of these produced fruit in any given year. We included in our study every wild tree from which we could obtain enough fruits, including trees from Parque Nacional Soberanía, Old Gamboa Road, Venta de Cruces, Parque Nacional Camino de Cruces, and Parque Natural Metropolitano . Sample sizes differ among variables and years and are provided below; our sample sizes increased from 2006 to 2008 as we found new individuals and added new sites. We defined trees as cultivated if they were obviously planted either alongside rural dwellings or as horticultural plantings in more urbanized areas. Whenever possible, we interviewed owners to obtain direct information about who planted the tree; we did not use trees whose origin was ambiguous. Chrysophyllum cainito is primarily propagated by seed, apparently exclusively so in Panama. We roughly matched the sample sizes and locations of our cultivated sample to the wild tree sample, 25 liter plant pot including trees from the communities of Gamboa, Chilibre, Chilibrillo, Paraíso, Ciudad del Saber, Los Rios, Burunga, and Balboa . From among the available trees, we chose our cultivated sample based primarily on access, as it was not always possible to get permission from owners to collect fruits.We collected five to thirty fruits per tree, and of these, five or three were randomly selected for analysis. We completed measurements on fruits almost always within several hours of collection, and always within three days. For each fruit, we took a photograph, noted color, measured fresh weight, length and diameter, and toughness using a penetrometer . We then cut the fruit open and measured the thickness of the exocarp, or rind . We extracted fluid from the fruit pulp by passing it through a fine filter and assessed sugar content with a hand-held refractometer . We counted the number of seeds and measured mean seed mass . One wild tree produced no healthy mature seeds, decreasing the sample size for seed mass to 28. Fruit shape is expressed as diameter/length, and proportion allocation to exocarp as /diameter.To compare wild and cultivated classes, we used nested ANOVA with trees nested within class , and samples nested within tree. Nested ANOVA also allowed us to test for significant variation among parent trees within the classes. We used Levene’s test to test for a difference between cultivated and wild trees in the variance of each trait. For any trait that showed heterogeneity of variance, P-values from the ANOVAwere suspect. Therefore, for the subset of traits that tested significant for Levene’s test, we performed Welch’s t-test assuming unequal variances on the comparison between wild and cultivated fruits—but this test could not be used to test for significant variance among trees within classes. Chemistry data from 2006 were analyzed separately from the 2008 data. While the geographical logistics of this system did not allow for the sampling of truly independent but comparable regions, our sample in 2008 was large enough to support separating the data into Northern and Southern sub-regions. When the analyses were done separately on these sub-regions, we obtained essentially the same patterns of difference between wild and cultivated classes for the various traits. Therefore, only the combined data are presented here. Pairwise correlation coefficients were calculated among traits in the 2008 sample and among chemical traits in the 2006 sample . Correlations between fruit and leaf phenolics could not be estimated because they came from different sets of trees. We used Discriminant Analysis to test whether the classes of wild and cultivated trees could be distinguished from each other, and what traits contribute to this discrimination. From the 2008 dataset, we included the following variables: Fruit mass, fruit diameter/length, exocarp proportion, toughness, seed number, seed mass, and sugar concentration . We left out the most redundant variables: Fruit length, diameter, and absolute exocarp width. We used forward stepwise variable selection to choose variables that significantly contributed to the model , then ran the model. For all trees in the sample that produced fruits both in 2007 and in 2008 , we calculated correlation coefficients between the mean trait value in 2007 and the mean trait value in 2008.We found many traits that differ significantly between wild and cultivated fruits of C. cainito. Fruits from cultivated genotypes were larger and had more pulp and less exocarp, resulting in a higher “economic ratio” sensu Clement . Fruit pulp had significantly higher concentrations of sugar, was less acidic, and had lower concentrations of phenolics. Seeds were bigger and more numerous and were less defended with phenolics in cultivated than in wild fruits. Historical accounts mention caimito already being cultivated in Panama in the mid-1500 s . Although these data are scant, we can infer that caimito has been utilized as a fruit crop for at least 450 years and possibly much longer. Caimito trees may come into bearing within about 15 years, allowing us to estimate that a minimum of 30 generations or possible rounds of human-mediated selection have occurred to date. Interestingly, early historical accounts mention the presence of large and small fruited types , suggesting that substantial selection may have already occurred by that time. The sugar concentration in C. cainito is extremely high. In 2008 we measured a mean °Brix of 14.9, with a maximum value of 28.