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Background and Aims At the population level, genetic diversity is a key determinant of a tree species’ capacity to cope with stress. However, little is known about the relative importance of the different components of genetic diversity for tree stress responses. We compared how two sources of genetic diversity, genotype and cytotype (i.e. differences in ploidy levels), influence growth, phytochemical and physiological traits of Populus tremuloides in the presence and absence of environmental stress. Methods In a series of field studies, we first assessed variation in traits across diploid and triploid aspen genotypes from Utah and Wisconsin under non-stressed conditions. In two follow-up experiments, we exposed diploid and triploid aspen genotypes from Wisconsin to individual and interactive drought stress and defoliation treatments and quantified trait variations under stress. Key Results We found that (1) tree growth and associated traits did not differ significantly between ploidy levels under non-stressed conditions. Instead, variation in tree growth and most other traits was driven by genotypic and population differences. (2) Genotypic differences were critical for explaining variation of most functional traits and their responses to stress. (3) Ploidy level played a subtle role in shaping traits and trait stress responses, as its influence was typically obscured by genotypic differences. (4) As an exception to the third conclusion, we showed that triploid trees expressed 17 % higher foliar defence (tremulacin) levels, 11 % higher photosynthesis levels and 23 % higher rubisco activity under well-watered conditions. Moreover, triploid trees displayed greater drought resilience than diploids as they produced 35 % more new tissue than diploids when recovering from drought stress. Conclusion Although ploidy level can strongly influence the ecology of tree species, those effects may be relatively small in contrast to the effects of genotypic variation in highly diverse species.

Plant resource allocation patterns often reveal tradeoffs that favor growth (G) over defense (D), or vice versa. Ecologists most often explain G-D tradeoffs through principles of economic optimality, in which negative trait correlations are attributed to the reconciliation of fitness costs. Recently, researchers in molecular biology have developed 'big data' resources including multi-omic (e.g. transcriptomic, proteomic and metabolomic) studies that describe the cellular processes controlling gene expression in model species. In this synthesis, we bridge ecological theory with discoveries in multi-omics biology to better understand how selection has shaped the mechanisms of G-D tradeoffs. Multi-omic studies reveal strategically coordinated patterns in resource allocation that are enabled by phytohormone crosstalk and transcriptional signal cascades. Coordinated resource allocation justifies the framework of optimality theory, while providing mechanistic insight into the feedbacks and control hubs that calibrate G-D tradeoff commitments. We use the existing literature to describe the coordinated resource allocation hypothesis (CoRAH) that accounts for balanced cellular controls during the expression of G-D tradeoffs, while sustaining stored resource pools to buffer the impacts of future stresses. The integrative mechanisms of the CoRAH unify the supply- and demand-side perspectives of previous G-D tradeoff theories.

Climate change is threatening the persistence of many tree species via independent and interactive effects on abiotic and biotic conditions. In addition, changes in temperature, precipitation, and insect attacks can alter the traits of these trees, disrupting communities and ecosystems. For foundation species such as Populus, phytochemical traits are key mechanisms linking trees with their environment and are likely jointly determined by interactive effects of genetic divergence and variable environments throughout their geographic range. Using reciprocal Fremont cottonwood (Populus fremontii) common gardens along a steep climatic gradient, we explored how environment (garden climate and simulated herbivore damage) and genetics (tree provenance and genotype) affect both foliar chemical traits and the plasticity of these traits. We found that (1) Constitutive and plastic chemical responses to changes in garden climate and damage varied among defense compounds, structural compounds, and leaf nitrogen. (2) For both defense and structural compounds, plastic responses to different garden climates depended on the climate in which a population or genotype originated. Specifically, trees originating from cool provenances showed higher defense plasticity in response to climate changes than trees from warmer provenances. (3) Trees from cool provenances growing in cool garden conditions expressed the lowest constitutive defense levels but the strongest induced (plastic) defenses in response to damage. (4) The combination of hot garden conditions and simulated herbivory switched the strategy used by these genotypes, increasing constitutive defenses but erasing the capacity for induction after damage. Because Fremont cottonwood chemistry plays a major role in shaping riparian communities and ecosystems, the effects of changes in phytochemical traits can be wide reaching. As the southwestern US is confronted with warming temperatures and insect outbreaks, these results improve our capacity to predict ecosystem consequences of climate change and inform selection of tree genotypes for conservation and restoration purposes., Global Change Biology, 28 (15), ISSN:1354-1013, ISSN:1365-2486

Climate warming can influence interactions between plants and associated organisms by altering levels of plant secondary metabolites. In contrast to studies of elevated temperature on aboveground phytochemistry, the consequences of warming on root chemistry have received little attention. Herein, we investigated the effects of elevated temperature, defoliation, and genotype on root biomass and phenolic compounds in trembling aspen (Populus tremuloides). We grew saplings of three aspen genotypes under ambient or elevated temperatures (+4–6 °C), and defoliated (by 75%) half of the trees in each treatment. After 4 months, we harvested roots and determined their condensed tannin and salicinoid (phenolic glycoside) concentrations. Defoliation reduced root biomass, with a slightly larger impact under elevated, relative to ambient, temperature. Elevated temperature decreased condensed tannin concentrations by 21–43% across the various treatment combinations. Warming alone did not alter salicinoid concentrations but eliminated a small negative impact of defoliation on those compounds. Graphical vector analysis suggests that effects of warming and defoliation on condensed tannins and salicinoids were predominantly due to reduced biosynthesis of these metabolites in roots, rather than to changes in root biomass. In general, genotypes did not differ in their responses to temperature or temperature by defoliation interactions. Collectively, our results suggest that future climate warming will alter root phytochemistry, and that effects will vary among different classes of secondary metabolites and be influenced by concurrent ecological interactions such as herbivory. Temperature- and herbivory-mediated changes in root chemistry have the potential to influence belowground trophic interactions and soil nutrient dynamics.

The ability to tolerate neighboring plants (i.e. degree of competitive response) is a key determinant of plant success in high-competition environments. Plant genotypes adjust their functional trait expression under high levels of competition, which may help explain intra-specific variation in competitive response. However, the relationships between traits and competitive response are not well understood, especially in trees. In this study, we investigated among-genotype associations between tree trait plasticity and competitive response. We manipulated competition intensity in experimental stands of trembling aspen (Populus tremuloides) to address the covariance between competition-induced changes in functional trait expression and aspects of competitive ability at the genotype level. Genotypic variation in the direction and magnitude of functional trait responses, especially those of crown foliar mass, phytochemistry, and leaf physiology, was associated with genotypic variation in competitive response. Traits exhibited distinct plastic responses to competition, with varying degrees of genotypic variation and covariance with other trait responses. The combination of genotypic diversity and covariance among functional traits led to tree responses to competition that were coordinated among traits yet variable among genotypes. Such relationships between tree traits and competitive success have the potential to shape stand-level trait distributions over space and time.

Plant chemistry plays an important role in mediating resistance against insect herbivores. Depending on insect species and feeding strategies, plant defenses can prevent insect attacks, impact digestive physiology and influence reproductive output. The Asian longhorned beetle (Anoplophora glabripennis) is an invasive, tree-killing insect that exploits multiple host plant species. Anoplophora glabripennis is documented to attack Populus spp., but the ability for adults to consume Populus varies between beetle populations. In this study, we explored the influence of salicinoid phenolic glycosides, known to be potent chemical defenses in Populus against generalist insects, on A. glabripennis adult behavior and fecundity. We performed a series of choice- and no-choice experiments using extracts of salicinoids applied to plants that do not produce these compounds. Salicinoids affected A. glabripennis performance in a dose-dependent manner, with higher concentrations significantly reducing feeding. Egg development was reduced by > 90% in females fed salicinoids at ~ 1.3% dry weight. We then compared salicinoids present in feeding material vs. frass and found both a decrease in concentration and a shift in composition of these compounds. However, some of the more complex salicinoids were unchanged in the excrement, suggesting minimal detoxification had occurred. Our results show that salicinoids disrupt egg development in A. glabripennis, thereby reducing fitness. These results have implications for how A. glabripennis metabolizes plant defense chemicals and how tolerance may vary among populations.

Several studies have demonstrated the ecological consequences of genetic variation within a single plant species. For example, these studies show that individual plant genotypes support unique composition of the plants' associated arthropod community. By contrast, fewer studies have explored how plant genetic variation may influence evolutionary dynamics in the plant's associated species. Here, we examine how aphids respond evolutionarily to genetic variation in their host plant. We conducted two experiments to examine local adaptation and rapid evolution of the free‐feeding aphid Chaitophorus populicola across genetic variants of its host plant, Populus angustifolia. To test for local adaptation, we collected tree cuttings and aphid colonies from three sites along an elevation/climate gradient and conducted a reciprocal transplant experiment. In general, home aphids (aphids transplanted onto trees from the same site) produced 1.7–3.4 times as many offspring as foreign aphids (aphids transplanted onto trees from different sites). To test for rapid evolution, we used 4 clonally replicated aphid genotypes and transplanted each onto 5 clonally replicated P. angustifolia genotypes. Each tree genotype started with the same aphid genotype composition. After 21 days (~two aphid generations), aphid genotype composition changed (i.e., aphids evolved) and some tree genotypes supported unique evolutionary trajectories of aphids. These results suggest that plant evolution in response to human perturbation, such as climate change and invasive species, will also result in evolutionary responses in strongly interacting species that could cascade to affect whole communities., We conducted two experiments to examine local adaptation and rapid evolution of the free‐feeding aphid Chaitophorus populicola across a genetic gradient of its host plant, Populus angustifolia. We found evidence of local adaptation, such that local aphids produced 1.7–3.4 times as many offspring as foreign aphids. Further, in just 21 days aphids rapidly and differentially evolved on different genotypes of their host plant.

Both environmental and genetic factors influence plant growth and foliar chemistry. The purpose of this experiment was to investigate the independent and interactive effects of soil community (and soil community diversity), tree genotype, and simulated browsing damage on the growth, biomass allocation and foliar chemistry of Populus tremuloides. We employed a factorial randomized complete block design with four soil treatments of varying diversity, five aspen genotypes, and a simulated ungulate browsing treatment. In the second year of growth, half the trees were subjected to simulated ungulate browsing, and 2 mo later trees were harvested and analyzed for biomass, biomass allocation, and foliar chemistry. Tree genotype was the strongest driver of foliar chemistry, but simulated browsing was the strongest driver of plant growth. Soil community influenced plant growth but not foliar chemistry, and soil community effects were rarely modified by tree genotype or simulated browsing. While environment and genotype had relatively equal influences on plant growth, tree genotype had a stronger influence than environmental effects (soil and simulated browsing) on foliar chemistry. We also documented negative plant–soil feedbacks for tree biomass. Our results highlight future opportunities for research on the potential effects of soil community diversity on trees.

Background and AimsIntraspecific variation in foundation species of forest ecosystems can shape community and ecosystem properties, particularly when that variation has a genetic basis. Traits mediating interactions with other species are predicted by simple allocation models to follow ontogenetic patterns that are rarely studied in trees. The aim of this research was to identify the roles of genotype, ontogeny and genotypic trade-offs shaping growth, defence and reproduction in aspen.MethodsWe established a common garden replicating >500 aspen genets in Wisconsin, USA. Trees were measured through the juvenile period into the onset of reproduction, for growth, defence chemistry (phenolic glycosides and condensed tannins), nitrogen, extrafloral nectaries, leaf morphology (specific leaf area), flower production and foliar herbivory and disease. We also assayed the TOZ19 sex marker and heterozygosity at ten microsatellite loci.Key ResultsWe found high levels of genotypic variation for all traits, and high heritabilities for both the traits and their ontogenetic trajectories. Ontogeny strongly shaped intraspecific variation, and trade-offs among growth, defence and reproduction supported some predictions while contradicting others. Both direct resistance (chemical defence) and indirect defence (extrafloral nectaries) declined during the juvenile stage, prior to the onset of reproduction. Reproduction was higher in trees that were larger, male and had higher individual heterozygosity. Growth was diminished by genotypic allocation to both direct and indirect defence as well as to reproduction, but we found no evidence of trade-offs between defence and reproduction.ConclusionsKey traits affecting the ecological communities of aspen have high levels of genotypic variation and heritability, strong patterns of ontogeny and clear trade-offs among growth, defence and reproduction. The architecture of aspen’s community genetics – its ontogeny, trade-offs and especially its great variability – is shaped by both its broad range and the diverse community of associates, and in turn further fosters that diversity.

Salicinoids form a specific class of phenolic glycosides characteristic of the Salicaceae. Although salicinoids accumulate in large amounts and have been shown to be involved in plant defense, their biosynthesis is unclear. We identified two sulfated salicinoids, salicin-7-sulfate and salirepin-7-sulfate, in black cottonwood (Populus trichocarpa). Both compounds accumulated in high amounts in above-ground tissues including leaves, petioles, and stems, but were also found at lower concentrations in roots. A survey of salicin-7-sulfate and salirepin-7-sulfate in a subset of poplar (Populus sp.) and willow (Salix sp.) species revealed a broader distribution within the Salicaceae. To elucidate the formation of these compounds, we studied the sulfotransferase (SOT) gene family in P. trichocarpa (PtSOT). One of the identified genes, PtSOT1, was shown to encode an enzyme able to convert salicin and salirepin into salicin-7-sulfate and salirepin-7-sulfate, respectively. The expression of PtSOT1 in different organs of P. trichocarpa matched the accumulation of sulfated salicinoids in planta. Moreover, RNA interference-mediated knockdown of SOT1 in gray poplar (Populus × canescens) resulted in decreased levels of sulfated salicinoids in comparison to wild-type plants, indicating that SOT1 is responsible for their formation in planta. The presence of a nonfunctional SOT1 allele in black poplar (Populus nigra) was shown to correlate with the absence of salicin-7-sulfate and salirepin-7-sulfate in this species. Food choice experiments with leaves from wild-type and SOT1 knockdown trees suggest that sulfated salicinoids do not affect the feeding preference of the generalist caterpillar Lymantria dispar. A potential role of the sulfated salicinoids in sulfur storage and homeostasis is discussed.

All organisms experience fundamental conflicts between divergent metabolic processes. In plants, a pivotal conflict occurs between allocation to growth, which accelerates resource acquisition, and to defense, which protects existing tissue against herbivory. Trade-offs between growth and defense traits are not universally observed, and a central prediction of plant evolutionary ecology is that context-dependence of these trade-offs contributes to the maintenance of intraspecific variation in defense [Zust and Agrawal, Annu. Rev. Plant Biol., 68, 513-534 (2017)]. This prediction has rarely been tested, however, and the evolutionary consequences of growth-defense trade-offs in different environments are poorly understood, especially in long-lived species [Cipollini et al., Annual Plant Reviews (Wiley, 2014), pp. 263-307]. Here we show that intraspecific trait trade-offs, even when fixed across divergent environments, interact with competition to drive natural selection of tree genotypes corresponding to their growth-defense phenotypes. Our results show that a functional trait trade-off, when coupled with environmental variation, causes real-time divergence in the genetic architecture of tree populations in an experimental setting. Specifically, competitive selection for faster growth resulted in dominance by fast-growing tree genotypes that were poorly defended against natural enemies. This outcome is a signature example of eco-evolutionary dynamics: Competitive interactions affected microevolutionary trajectories on a timescale relevant to subsequent ecological interactions [Brunner et al., Funct. Ecol. 33, 7-12 (2019)]. Eco-evolutionary drivers of tree growth and defense are thus critical to stand-level trait variation, which structures communities and ecosystems over expansive spatiotemporal scales.

Polyploidy, the expression of more than two sets of chromosomes, is common in plants, and is thought to influence plant trait expression and drive plant species evolution. The degree to which polyploidy influences interactions among physiological processes such as growth and defense in natural populations through its effect on phenotypic variability is poorly understood. We link broad plant genotypic features (including polyploidy) to phenotypic expression of growth and chemical defense in natural populations of quaking aspen (Populus tremuloides) to examine patterns in resource allocation that might drive growth-defense tradeoffs. Quaking aspen are capable of rapid growth, and are also a primary food plant for a large range of herbivores, including insects and ungulates. While often diploid, aspen can exhibit polyploidy as triploid clones. We tested for the effect of genotype, cytotype (ploidy level, divided between diploids and triploids), and ramet age on relationships between growth and leaf chemistry across natural aspen clones in northern Utah. Substantial genotype variability in growth and leaf chemistry occurred across both cytotypes. Phenolic glycosides, but not condensed tannins, were negatively related to growth. Ramet age was also negatively related to growth. Phenolic glycosides were negatively related to condensed tannins, but only for the diploid clones. Triploid clones exhibited ~ 20% higher levels of phenolic glycosides than diploids. Growth in quaking aspen was likely sacrificed for the production of phenolic glycosides. Our study underscores the importance of considering polyploidy, genetic variability, and ramet age in understanding growth-defense tradeoffs in natural populations of clonal organisms, such as quaking aspen.

Patterns of trait expression within some plant species have recently been shown to follow patterns described by the leaf economics spectrum paradigm. Resistance to herbivores is also expected to covary with leaf economics traits. We selected multiple mature Populus tremuloides genotypes from a common garden to assess whether aspen leaf economics patterns follow those observed among species globally. We also evaluated leaf economics strategies in the context of insect resistance by conducting bioassays to determine the effects of plant traits on preference and performance of Lymantria dispar. We found that: 1) intraspecific trait patterns of P. tremuloides parallel those exhibited by the interspecific leaf economics spectrum, 2) herbivores preferred leaves from genotypes with resource-acquisitive strategies, and 3) herbivores also performed best on genotypes with resource-acquisitive strategies. We conclude that a leaf economics spectrum that incorporates defense traits is a useful tool for explaining intraspecific patterns of variation in plant strategies, including resistance to herbivores.

The North American beaver (Castor canadensis Kuhl) and cottonwoods (Populus spp.) are foundation species, the interactions of which define a much larger community and affect a threatened riparian habitat type. Few studies have tested the effect of these interactions on plant chemistry and a diverse arthropod community. We experimentally examined the impact of beaver foraging on riparian communities by first investigating beaver food preferences for one cottonwood species, Fremont cottonwood (P. fremontii S. Watson), compared to other locally available woody species. We next examined the impact of beaver foraging on twig chemistry and arthropod communities in paired samples of felled and unfelled cottonwood species in northern Arizona (P. fremontii) and southwestern Colorado (narrowleaf cottonwood, P. angustifolia James, and Eastern cottonwood, P. deltoides W. Bartram ex Marshall). Four major patterns emerged: (1) In a cafeteria experiment, beavers chose P. fremontii six times more often than other woody native and exotic species. (2) With two cottonwood species, we found that the nitrogen and salicortin concentrations were up to 45% greater and lignin concentration 14% lower in the juvenile resprout growth of felled trees than the juvenile growth on unfelled trees (six of seven analyses were significant for P. fremontii and four of six were significant for P. angustifolia). (3) With two cottonwood species, arthropod community composition on juvenile branches differed significantly between felled and unfelled trees, with up to 38% greater species richness, 114% greater relative abundance and 1282% greater species diversity on felled trees (six of seven analyses with P. fremontii and four of six analyses with P. angustifolia were significant). The above findings indicate that the highest arthropod diversity is achieved in the heterogenous stands of mixed felled and unfelled trees than in stands of cottonwoods, where beavers are not present. These results also indicate that beaver herbivory changes the chemical composition in 10 out of 13 chemical traits in the juvenile growth of two of the three cottonwood species to potentially allow better defense against future beaver herbivory. (4) With P. deltoides, only one of five analyses in chemistry was significant, and none of the four arthropod community analyses were significant, suggesting that this species and its arthropod community responds differently to beaver. Potential reasons for these differences are unknown. Overall, our findings suggest that in addition to their impact on riparian vegetation, other mammals, birds, and aquatic organisms, beavers also may define the arthropod communities of two of three foundation tree species in these riparian ecosystems.

Patterns of trait expression within some plant species have recently been shown to align with the leaf economics spectrum paradigm. Resistance to herbivores is also expected to covary with leaf economics traits. We selected 36 mature Populus tremuloides genotypes in a common garden to assess whether aspen leaf economics patterns follow those observed among species globally. We also evaluated leaf economics strategies in the context of insect resistance by conducting bioassays to determine the effects of plant traits on preference and performance of Lymantria dispar. We found that: (1) intraspecific trait patterns of P. tremuloides parallel those exhibited by the interspecific leaf economics spectrum, (2) herbivores preferred leaves from genotypes with resource-acquisitive strategies, and (3) herbivores also performed best on genotypes with resource-acquisitive strategies. We conclude that a leaf economics spectrum that incorporates defense traits is a useful tool for explaining intraspecific patterns of variation in plant strategies, including resistance to herbivores.

Although genetic diversity within stands of trees is known to have community-level consequences, whether such effects are present at an even finer genetic scale is unknown. We examined the hypothesis that genetic variability (heterozygosity) within an individual plant would affect its dependent community, which adds a new dimension to the importance of genetic diversity. Our study contrasted foliar arthropod community diversity and microsatellite marker-derived measures of genetic diversity of cottonwood (Populus fremontii) trees that had been felled by beavers (Castor canadensis) and were resprouting, relative to adjacent standing, unfelled trees. Three patterns emerged: 1. Productivity (specific leaf area), phytochemical defenses (salicortin), and arthropod community richness, abundance, and diversity were positively correlated with the heterozygosity of individual felled trees, but not with that of unfelled trees, 2. These relationships were not explained by population substructure, genetic relatedness of the trees, or hybridization, 3. The underlying mechanism appears to be that beaver herbivory stimulates increased productivity (i.e., 2× increase from the most homozygous to the most heterozygous tree) that is the greatest in more heterozygous trees. Salicortin defenses in twigs were also expressed at higher concentrations in more heterozygous trees (i.e., 3× increase from the most homozygous to the most heterozygous tree), which suggests that this compound may dissuade further herbivory by beavers, as has been found for other mammalian herbivores. We suggest that high stress to trees as a consequence of felling reveals a heterozygosity–productivity linkage, which in turn is attractive to arthropods. Although experiments are required to demonstrate causality, these results link the genetic diversity of individual trees to community diversity, supporting the hypothesis that interactions among foundation species (beavers and trees) have community-level effects, and underscores the importance of genetic diversity for biodiversity, conservation, and restoration.

Efforts to maintain the function of critical ecosystems under climate change often begin with foundation species. In the southwestern United States, cottonwood trees support diverse communities in riparian ecosystems that are threatened by rising temperatures. Genetic variation within cottonwoods shapes communities and ecosystems, but these effects may be modified by phenotypic plasticity, where genotype traits change in response to environmental conditions. Here, we investigated plasticity in Fremont cottonwood (Populus fremontii) leaf litter traits as well as the consequences of plasticity for riparian ecosystems. We used three common gardens each planted with genotypes from six genetically divergent populations spanning a 12°C temperature gradient, and a decomposition experiment in a common stream environment. We found that leaf litter area, specific leaf area, and carbon to nitrogen ratio (C:N) were determined by interactions between genetics and growing environment, as was the subsequent rate of litter decomposition. Most of the genetic variation in leaf litter traits appeared among rather than within source populations with distinct climate histories. Source populations from hotter climates generally produced litter that decomposed more quickly, but plasticity varied the magnitude of this effect. We also found that hotter growing conditions reduced the variation in litter traits produced across genotypes, homogenizing the litter inputs to riparian ecosystems. All genotypes in the hottest garden produced comparatively small leaves that decomposed quickly and supported lower abundances of aquatic invertebrates, whereas the same genotypes in the coldest garden produced litter with distinct morphologies and decomposition rates. Our results suggest that plastic responses to climate stress may constrict the expression of genetic variation in predictable ways that impact communities and ecosystems. Understanding these interactions between genetic and environmental variation is critical to our ability to plan for the role of foundation species when managing and restoring riparian ecosystems in a warming world.

Climate warming can influence interactions between plants and associated organisms by altering levels of plant secondary metabolites. In contrast to studies of elevated temperature on aboveground phytochemistry, the consequences of warming on root chemistry have received little attention. Herein, we investigated the effects of elevated temperature, defoliation, and genotype on root biomass and phenolic compounds in trembling aspen (Populus tremuloides). We grew saplings of three aspen genotypes under ambient or elevated temperatures (+4-6 °C), and defoliated (by 75%) half of the trees in each treatment. After 4 months, we harvested roots and determined their condensed tannin and salicinoid (phenolic glycoside) concentrations. Defoliation reduced root biomass, with a slightly larger impact under elevated, relative to ambient, temperature. Elevated temperature decreased condensed tannin concentrations by 21-43% across the various treatment combinations. Warming alone did not alter salicinoid concentrations but eliminated a small negative impact of defoliation on those compounds. Graphical vector analysis suggests that effects of warming and defoliation on condensed tannins and salicinoids were predominantly due to reduced biosynthesis of these metabolites in roots, rather than to changes in root biomass. In general, genotypes did not differ in their responses to temperature or temperature by defoliation interactions. Collectively, our results suggest that future climate warming will alter root phytochemistry, and that effects will vary among different classes of secondary metabolites and be influenced by concurrent ecological interactions such as herbivory. Temperature- and herbivory-mediated changes in root chemistry have the potential to influence belowground trophic interactions and soil nutrient dynamics.

INTRODUCTION Condensed tannins (CTs) are proanthocyanidin heteropolymers that are widely distributed among plants. Their biochemical properties are determined by molecular structure (e.g. polymer size, hydroxylation, stereochemistry). In Populus, genetically and environmentally-determined CT concentrations have been related to ecological effects, while the potential role of CT molecular structure has received little attention. OBJECTIVE Evaluate CT polymerisation, major constituent monomers, stereochemistry and overall content in Populus tremuloides foliage using ultra-high-performance liquid chromatography with photodiode array and mass spectrometry (UPLC-PDA-(-)esi-MS) detection following thiolytic depolymerisation of the CTs. METHODOLOGY CTs were extracted from dried foliage of six P. tremuloides genotypes into methanol and thiolytically depolymerised into constituent monomers. Calibration standards were prepared by thiolysis of CT mixtures isolated from P. tremuloides foliage on Sephadex LH-20, followed by preparative high-performance liquid chromatography (HPLC). RESULTS Populus tremuloides CTs contained predominantly repeating subunits of three putative stereoisomers each of catechin and gallocatechin. Linear calibrations for standards of these subunits and their thioethers (purities 44-87%, UPLC-(-)esi-MS) were generally stable over the course of 10 months. CT polymer size, hydroxylation, stereochemistry and concentrations differed among genotypes. CONCLUSION This thiolysis-UPLC-PDA-(-)esiMS method was optimised for analysis of CT polymer size, hydroxylation, stereochemistry, and total concentration in Populus foliage. It revealed significant variation in each of these properties among P. tremuloides genotypes, and will facilitate evaluation of how environmental factors affect CT molecular structures.

Empirical studies indicate that host-tree bud break will likely advance faster than spring-folivore egg hatch in response to predicted increases in temperature. How these phenological shifts will affect herbivory will depend on temporal patterns of foliar traits that occur during leaf expansion, and their effects on folivore performance. Through fine-scale time series sampling of newly flushed trembling aspen (Populus tremuloides) foliage, we observed a previously unknown peak in phenolic glycoside concentrations that coincides with the emergence of sensitive neonates of gypsy moths and rapidly declines soon after bud break. The magnitude and duration of the initial post-bud break peak in phenolic glycosides varied substantially among genotypes. In contrast, foliar nitrogen concentrations declined at a more uniform rate among genotypes throughout leaf expansion. In addition, leaf toughness remained uniformly low throughout these periods of phytochemical change, and did not rise or vary substantially among genotypes until after anticipated windows of climate change-induced shifts between bud break and egg hatch had elapsed. Controlled manipulation of intervals between gypsy moth egg hatch and aspen bud break generated differences in larval performance among hatch cohorts and host genotypes that corresponded with changes in foliar phenolic glycoside and nitrogen concentrations. These findings indicate that the effects of climate change-induced phenological shifts on herbivory will differ among host plant genotypes, and that genetic variation in foliar chemical patterns will strongly influence this heterogeneity.

Genomic studies have been used to identify genes underlying many important plant secondary metabolic pathways. However, genes for salicinoid phenolic glycosides (SPGs)—ecologically important compounds with significant commercial, cultural, and medicinal applications—remain largely undescribed. We used a linkage map derived from a full‐sib population of hybrid cottonwoods (Populus spp.) to search for quantitative trait loci (QTL) for the SPGs salicortin and HCH‐salicortin. SSR markers and primer sequences were used to anchor the map to the V3.0 P. trichocarpa genome. We discovered 21 QTL for the two traits, including a major QTL for HCH‐salicortin (R 2 = .52) that colocated with a QTL for salicortin on chr12. Using the V3.0 Populus genome sequence, we identified 2,983 annotated genes and 1,480 genes of unknown function within our QTL intervals. We note ten candidate genes of interest, including a BAHD‐type acyltransferase that has been potentially linked to Populus SPGs. Our results complement other recent studies in Populus with implications for gene discovery and the evolution of defensive chemistry in a model genus. To our knowledge, this is the first study to use a full‐sib mapping population to identify QTL intervals and gene lists associated with SPGs.

Plant stature can be strongly modified via regulation of endogenous levels and signalling of the plant hormone gibberellin (GA). Down-regulation of GA can produce semi-dwarf tree varieties with improved qualities such as reduced susceptibility to wind damage, enhanced root growth and more compact cultivation. However, these modifications may have unintended, non-target consequences for defence against herbivores, via either of two mechanisms: (i) reduced biomass production may cause trade-offs with chemical resistance traits, as predicted by the growth-differentiation balance hypothesis, and (ii) altered biomass allocation to either roots or photosynthetic tissues may affect regrowth potential and thus tolerance to defoliation. We studied GA down-regulated (GE) and non-transgenic wild-type hybrid poplar (Populus alba × P. tremula) in an outdoor, aboveground common garden and defoliated half of all replicate trees to simulate defoliation. We then quantified the independent and interactive effects of genotype and defoliation on growth and chemical resistance-related traits, including phenolic glycosides (PGs), condensed tannin and nitrogen. We also calculated tolerance to defoliation as the differential in relative growth between undefoliated and defoliated trees. Our results indicate that two of the four GA down-regulated genotypes had significantly reduced stem height, basal diameter, volume (d²h), total biomass and increased allocation to leaves relative to the wild type. One of those two genotypes also had reduced allocation to roots. One and sometimes both of these same two genotypes also had at least 20% lower levels of condensed tannins and PGs and similar increases in lignin and nitrogen. Tolerance, as calculated by the differential in relative growth between undefoliated and defoliated trees, was similar among all experimental genotypes. However, two GE genotypes flushed fewer leaves in response to defoliation relative to the wild type. Our results indicate that GA down-regulation strongly alters biomass production and allocation in poplar but does not necessarily compromise the ability of these trees to tolerate damage. However, some of the modifications we observed do have the potential to alter non-target resistance traits over time, and warrant further research, especially under plantation conditions.

Although chemical deterrents to herbivory often exact costs in terms of plant growth, the manner in which those costs arise, and their physiological relationship to other functional traits, remain unclear. In the absence of appreciable herbivory, we examined interrelationships among chemical defense levels and other foliar functional traits (e.g., light-saturated photosynthesis, specific leaf area, nitrogen concentration) as co-determinants of tree growth and, by extension, competitive ability in high-density populations comprising 16 genotypes of Populus tremuloides. Across genotypes, concentrations of chemical defenses were not significantly related to other leaf functional traits, but levels of the salicinoid phenolic glycosides (SPGs) salicin, salicortin and tremulacin were each negatively correlated with relative mass growth (RMG) of aboveground woody tissue (P ≤ 0.001). RMG, in turn, underpinned 77% of the genotypic variation in relative height growth (our index of competitive ability). RMG was also positively related to light-saturated photosynthesis (P ≤ 0.001), which, together with the three SPGs, explained 86% of genotypic RMG variation (P ≤ 0.001). Moreover, results of a carbon balance simulation indicated that costs of resource allocation to SPGs, reaching nearly a third of annual crown photosynthesis, were likely mediated by substantial metabolic turnover, particularly for salicin. The lack of discernible links between foliar defense allocation and other (measured) functional traits, and the illustrated potential of metabolic turnover to reconcile influences of SPG allocation on RMG, shed additional light on fundamental physiological mechanisms underlying evolutionary tradeoffs between chemical defense investment and competitive ability in a foundation tree species.

Community genetics aims to understand the effects of intraspecific genetic variation on community composition and diversity, thereby connecting community ecology with evolutionary biology. Thus far, research has shown that plant genetics can underlie variation in the composition of associated communities (e.g., insects, lichen and endophytes), and those communities can therefore be considered as extended phenotypes. This work, however, has been conducted primarily at the plant genotype level and has not identified the key underlying genes. To address this gap, we used genome-wide association mapping with a population of 445 aspen (Populus tremuloides) genets to identify the genes governing variation in plant traits (defence chemistry, bud phenology, leaf morphology, growth) and insect community composition. We found 49 significant SNP associations in 13 Populus genes that are correlated with chemical defence compounds and insect community traits. Most notably, we identified an early nodulin-like protein that was associated with insect community diversity and the abundance of interacting foundation species (ants and aphids). These findings support the concept that particular plant traits are the mechanistic link between plant genes and the composition of associated insect communities. In putting the "genes" into "genes to ecosystems ecology", this work enhances understanding of the molecular genetic mechanisms that underlie plant-insect associations and the consequences thereof for the structure of ecological communities.

Anthropogenic activities are altering levels of atmospheric carbon dioxide (CO2) and tropospheric ozone (O3). These changes can alter phytochemistry, and in turn, influence ecosystem processes. We assessed the individual and combined effects of elevated CO2 and O3 on the phytochemical composition of two tree species common to early successional, northern temperate forests. Trembling aspen (Populus tremuloides) and paper birch (Betula papyrifera) were grown at the Aspen FACE (Free-Air Carbon dioxide and ozone Enrichment) facility under four combinations of ambient and elevated CO2 and O3. We measured, over three years (2006-08), the effects of CO2 and O3 on a suite of foliar traits known to influence forest functioning. Elevated CO2 had minimal effect on foliar nitrogen and carbohydrate levels in either tree species, and increased synthesis of condensed tannins and fiber in aspen, but not birch. Elevated O3 decreased nitrogen levels in both tree species and increased production of sugar, condensed tannins, fiber, and lignin in aspen, but not birch. The magnitude of responses to elevated CO2 and O3 varied seasonally for both tree species. When co-occurring, CO2 offset most of the changes in foliar chemistry expressed under elevated O3 alone. Our results suggest that levels of CO2 and O3 predicted for the mid-twenty-first century will alter the foliar chemistry of northern temperate forests with likely consequences for forest community and ecosystem dynamics.

Diseases can markedly alter the ecological and economic value of poplars. To better understand poplar–pathogen interactions, we investigated the independent and interactive effects of tree genotype, soil nutrient limitation, and interspecific competition on incidence of powdery mildew (caused by the fungal obligate pathogen Erysiphe adunca (Wallr.) Fr., 1829) in a foundation tree species, trembling aspen (Populus tremuloides Michx.). We established a common garden of potted aspen saplings, incorporating five tree genotypes, two levels of soil nutrients (low and high), and two levels of competition (with and without grass). We then surveyed natural incidence of powdery mildew and aspen vigor (i.e., growth). Incidence of powdery mildew varied among aspen genotypes, and variance in incidence shifted among environments in which the trees were grown. Added soil nutrients increased powdery mildew incidence on aspen, whereas grass competition had the opposite effect. Interestingly, grass competition either enhanced or dampened the variance in incidence of powdery mildew among tree genotypes, depending on soil nutrient levels. In addition, powdery mildew incidence was positively related to tree vigor. Our findings reveal strong genetic, environmental, and genetic×environmental effects of disease on a foundation tree species and that particular environments can either enhance or diminish variation in responses among tree genotypes.

Animals have several strategies to contend with nutritionally poor diets, including compensatory consumption and enhanced food utilization efficiencies. Plants produce a diversity of defense compounds that affect the ability of herbivores to utilize these strategies in response to variation in food nutritional quality. Little is known, however, about effects of allelochemicals on herbivores utilizing integrated behavioral and morphological responses to reduced food quality. Our objectives were to (1) examine how variation in diet nutritional quality influences compensatory responses of a generalist insect herbivore, and (2) determine how plant defenses affect these processes. Gypsy moth (Lymantria dispar) larvae were administered one of nine combinations of diet having low, moderate, or high nutritional quality and 0, 2, or 4 % purified aspen (Populus tremuloides) salicinoids. We quantified larval growth, consumption, frass production, and biomass allocation to midgut tissue over a 4-day bioassay. In the absence of salicinoids, larvae compensated for reduced nutritional quality and maintained similar growth across all diets through increased consumption, altered midgut biomass allocation, and improved processing efficiencies. Dietary salicinoids reduced larval consumption, midgut biomass allocation, digestive efficiencies, and growth at all nutritional levels, but the effect size was more pronounced when larvae were fed nutritionally suboptimal diets. Our findings demonstrate that integrated behavioral and morphological compensatory responses to reduced food quality are affected by plant defenses, ultimately limiting compensatory responses and reducing larval performance.

Summary 1.Overbrowsing by ungulates is a major cause of poor aspen stand regeneration across North America and Eurasia. In general, factors driving ungulate browser preferences include concentrations of plant secondary compounds and the nutritional composition (non-structural carbohydrates, protein, and minerals) of foliage. While each of these phytochemical factors has been shown to independently influence ungulate preference, the relative impact of each factor is unknown, as no study to date has examined them simultaneously. 2.Plant fitness depends not only on the capacity of plants to resist browsing, but also on their capacity to tolerate browsing once it has occurred. Little is known of aspen tolerance to browsing, which inflicts a different form of damage than insect herbivory. 3.We employed multiple aspen genotypes, replicate trees of which were subjected to different soil nutrient treatments, to investigate: 1) the effects of aspen genotype, nutrient treatment, and genotype x nutrient interactions on susceptibility to browsing by white-tailed deer, 2) the phytochemical basis for the patterns observed in (1), and 3) the effects of genotype, soil nutrients, and their interaction on short-term tolerance to deer browsing. 4.Aspen genotypes varied markedly in their vernal susceptibility to deer browsing. Genetic variation in early season levels of non-structural carbohydrates (sugars), protein, and multiple macro- and trace minerals had the strongest influence on tree susceptibility to browsing. In contrast, levels of phytochemical defenses had minimal effects, although the range of levels expressed in this study was small. Soil nutrient availability did not significantly influence deer preference. 5.The extent of browsing affected post-browse tolerance across genotypes. Soil nutrient treatment had little differential effect on tolerance, and, for the most part, genotypes did not display differential tolerance to browsing, regardless of which soil nutrient treatment they experienced. 6.Synthesis: Genetic variation for susceptibility to browsing indicates that ungulate browsers have the potential to be agents of selection in aspen populations. In contrast with previous studies in aspen highlighting the importance of phytochemical defenses in shaping preferences of browsing mammals, our results indicate that the nutritional composition of foliage (sugars, protein, and mineral concentrations) can have sizable effects on preference. The observed lack of influence of soil nutrient availability on tree browsing tolerance contrasts with predictions of the limiting resource model, the prevailing model for plant tolerance. This article is protected by copyright. All rights reserved.

Although plant growth is generally recognized to be influenced by allocation to defense, genetic background (e.g., inbreeding), and gender, rarely have those factors been addressed collectively. In quaking aspen (Populus tremuloides Michx.), phenolic glycosides (PGs) and condensed tannins (CTs) constitute up to 30 % of leaf dry weight. To quantify the allocation cost of this chemical defense, we measured growth, defense chemistry, and individual heterozygosity (H obs at 16 microsatellite loci) for male and female trees in both controlled and natural environments. The controlled environment consisted of 12 juvenile genets grown for 3 years in a common garden, with replication. The natural environment consisted of 51 mature genets in wild populations, from which we sampled multiple ramets (trees) per genet. Concentrations of PGs and CTs were negatively correlated. PGs were uncorrelated with growth, but CT production represented a major cost. Across the range of CT levels found in wild-grown trees, growth rates varied by 2.6-fold, such that a 10 % increase in CT concentration occurred with a 38.5 % decrease in growth. H obs had a marked effect on aspen growth: for wild trees, a 10 % increase in H obs corresponded to a 12.5 % increase in growth. In wild trees, this CT effect was significant only in females, in which reproduction seems to exacerbate the cost of defense, while the H obs effect was significant only in males. Despite the lower growth rate of low-H obs trees, their higher CT levels may improve survival, which could account for the deficit of heterozygotes repeatedly found in natural aspen populations.

Global climate change has the potential to dramatically alter multiple ecosystem processes, including herbivory. The development rates of both plants and insects are highly sensitive to temperature. Although considerable work has examined the effects of temperature on spring phenologies of plants and insects individually, few studies have examined how anticipated warming will influence their phenological synchrony. We applied elevated temperatures of 1.7 and 3.4 °C in a controlled chamberless outdoor experiment in northeastern Minnesota, USA to examine the relative responses in onset of egg eclosion by forest tent caterpillar (Malacosoma disstria Hubner) and budbreak of two of its major host trees (trembling aspen, Populus tremuloides Michaux, and paper birch, Betula papyrifera Marshall). We superimposed four insect population sources and two overwintering regimes onto these treatments, and computed degree-day models. Timing of egg hatch varied among population source, overwintering location, and spring temperature regime. As expected, the development rates of plants and insects advanced under warmer conditions relative to ambient controls. However, budbreak advanced more than egg hatch. The degree of phenological synchrony between M. disstria and each host plant was differentially altered in response to warming. The interval by which birch budbreak preceded egg hatch nearly doubled from ambient to +1.7 °C. In the case of aspen, the sequence changed from egg hatch preceding, to following, budbreak at +3.4 °C. Additionally, under temperature regimes simulating future conditions, some insect populations currently south of our study sites became more synchronous with the manipulated hosts than did currently coexisting insect populations. These findings reveal how climate warming can alter insect-host plant interactions, through changes in phenological synchrony, possibly driving host shifts among tree species and genotypes. They also suggest how herbivore variability, both among populations and within individual egg masses, may provide opportunities for adaptation, especially in species that are highly mobile and polyphagous.

Many spring-feeding insect folivores have evolved to hatch in synchrony with temporal windows of high foliar quality that occur during leaf expansion. In some host-tree species, however, budbreak phenology can shift in response to defoliation during the prior growing season. While this response could have important impacts on the performance of insect folivore species prone to multi-year outbreaks, the prevalence, underlying mechanisms, and ecological ramifications of defoliation-induced phenological shifts are poorly understood. We established a common garden of twelve trembling aspen (Populus tremuloides Michx.) genotypes, and observed the effects of prior-season defoliation and soil-nutrient availability on budbreak phenology during the following spring. Average budbreak phenology was delayed in response to both prior-season defoliation and low soil-nutrient treatments, but these responses were lower in magnitude than those previously shown to occur in response to temperature. Magnitudes of budbreak delay varied significantly among genotypes and were highest among early-flushing aspen. This negative correlation between aspen budbreak phenology and magnitudes of defoliation-induced phenological delay suggests that widespread defoliation could decrease intraspecific variation in aspen budbreak phenology during subsequent growing seasons, potentially allowing larvae to adapt to windows of optimal foliage quality during multi-year outbreaks. These results quantify additional environmental factors that may act in concert with major drivers such as temperature to mediate phenological relationships between aspen and folivorous insects.

Induction of plant chemical defenses in response to insect feeding may be localized to the site of damage or expressed systemically, mediated by signal transduction throughout the plant. Such systemic induction processes have been widely investigated in plants with single stems, but rarely in clonal plants comprised of multiple ramets with vascular connections. For a clonal tree species such as trembling aspen (Populus tremuloides Michx), integration of induced defense within clones could be adaptive, as clones are spatially extensive and susceptible to outbreak herbivores. We used pairs of aspen saplings with shared roots, replicated from three genotypes, to determine whether defense-induction signals are communicated within clones. One ramet in each pair was subjected to a damage treatment (feeding by Lymantria dispar, followed by mechanical damage), and subsequent changes in leaf defensive chemistry were measured in both ramets. Responses to damage varied by defense type: condensed tannins (CTs) increased in damaged ramets but not in connected undamaged ramets, whereas salicinoid phenolic glycosides (SPGs) were not induced in any ramets. Genotypes varied in their levels of CTs, but not in their levels of SPGs, and responded similarly to damage treatment. These results suggest that, even with both vascular and volatile information available, young aspen ramets do not induce defenses based on signals or metabolites from other ramets. Thus, unlike other clonal plant species, aspen do not appear to coordinate defense induction within clones. Lack of coordinated early induction in aspen may be related to the function of CTs in tolerance, rather than resistance.

The accumulation of ultraviolet (UV)-absorbing compounds (flavonoids and related phenylpropanoids) and the resultant decrease in epidermal UV transmittance (TUV ) are primary protective mechanisms employed by plants against potentially damaging solar UV radiation and are critical components of the overall acclimation response of plants to changing solar UV environments. Whether plants can adjust this UV sunscreen protection in response to rapid changes in UV, as occurs on a diurnal basis, is largely unexplored. Here, we use a combination of approaches to demonstrate that plants can modulate their UV-screening properties within minutes to hours, and these changes are driven, in part, by UV radiation. For the cultivated species Abelmoschus esculentus, large (30-50%) and reversible changes in TUV occurred on a diurnal basis, and these adjustments were associated with changes in the concentrations of whole-leaf UV-absorbing compounds and several quercetin glycosides. Similar results were found for two other species (Vicia faba and Solanum lycopersicum), but no such changes were detected in Zea mays. These findings reveal a much more dynamic UV-protection mechanism than previously recognized, raise important questions concerning the costs and benefits of UV-protection strategies in plants and have practical implications for employing UV to enhance crop vigor and quality in controlled environments.

Numerous studies have explored the impacts of intraspecific genetic variation and environment on the induction of plant chemical defenses by herbivory. Relatively few, however, have considered how those factors affect within-plant distribution of induced defenses. This work examined the impacts of plant genotype and soil nutrients on the local and systemic phytochemical responses of trembling aspen (Populus tremuloides) to defoliation by gypsy moth (Lymantria dispar). We deployed larvae onto foliage on individual tree branches for 15 days and then measured chemistry in leaves from: 1) branches receiving damage, 2) undamaged branches of insect-damaged trees, and 3) branches of undamaged control trees. The relationship between post-herbivory phytochemical variation and insect performance also was examined. Plant genotype, soil nutrients, and damage all influenced phytochemistry, with genotype and soil nutrients being stronger determinants than damage. Generally, insect damage decreased foliar nitrogen, increased levels of salicinoids and condensed tannins, but had little effect on levels of a Kunitz trypsin inhibitor, TI3. The largest damage-mediated tannin increases occurred in leaves on branches receiving damage, whereas the largest salicinoid increases occurred in leaves of adjacent, undamaged branches. Foliar nitrogen and the salicinoid tremulacin had the strongest positive and negative relationships, respectively, with insect growth. Overall, plant genetics and environment concomitantly influenced both local and systemic phytochemical responses to herbivory. These findings suggest that herbivory can contribute to phytochemical heterogeneity in aspen foliage, which may in turn influence future patterns of herbivory and nutrient cycling over larger spatial scales.

While the importance of plant secondary metabolites to belowground functioning is gaining recognition, the perception remains that secondary metabolites are produced for herbivore defense, whereas their belowground impacts are ecological by-products, or 'afterlife' effects. However, plants invest a significant amount of resources into production of secondary metabolites that have minimal effects on herbivore resistance (e.g. condensed tannins and insect herbivores). We show that genetically mediated variation in condensed tannin concentration is correlated with plant nitrogen recovery following a severe defoliation event. We used single-tree mesocosms labeled with (15) N to track nitrogen through both the frass and litter cycling pathways. High concentrations of leaf tannins in Populus tremuloides were correlated with (15) N recovery from frass within the same growing season and in the following growing season. Likewise, leaf tannin concentrations were also correlated with (15) N recovery from the litter of defoliated trees in the growing season following the defoliation event. Conversely, tannins were not well correlated with nitrogen uptake under conditions of nominal herbivory. Our results suggest that tannins may confer benefits in response to herbivore pressure through conserved belowground nitrogen cycling, rather than via defensive properties. Consequently, tannins may be considered as chemical mediators of tolerance rather than resistance.