Temperature alters the toxicological impacts of plant terpenoids on the polyphagous model herbivore Vanessa cardui.

Terpenes are a major class of secondary metabolites present in all plants, and long hypothesized to have diversified in response to specific plant-herbivore interactions. Herbivory is a major biotic interaction that plays out across broad temporal and spatial scales that vary dramatically in temperature regimes, both due to climatic variation across geographic locations as well as the effect of seasonality. In addition, there is an emerging understanding that global climate change will continue to alter the temperature regimes of nearly every habitat on Earth over the coming centuries. Regardless of source, variation in temperature may influence herbivory, in particular via changes in the efficacy and impacts of plant defensive chemistry. This study aims to characterize temperature-driven variation in toxicological effects across several structural classes of terpenes in the model herbivore Vanessa cardui, the painted lady butterfly. We observed a general increase in monoterpene toxicity to larvae, pupa, and adults at higher temperatures, as well as an increase in development time as terpene concentration increased. Results obtained from this study yield insights into possible drivers of seasonal variation in plant terpene production as well as inform effects of rising global temperatures on plant-insect interactions. In the context of other known effects of climate change on plant-herbivore interactions like carbon fertilization and compensatory feeding, temperature-driven changes in plant chemical defense efficacy may further complicate the prediction of climate change impacts on the fundamental ecological process of herbivory.

Intraspecific variation in mycorrhizal response is much larger than ecological literature suggests.

Mycorrhizal response is the most common metric for characterizing how much benefit a plant derives from mycorrhizal symbiosis. Traditionally, ecologists have used these metrics to generalize benefit from mycorrhizal symbiosis in plant species, ignoring the potential for plant intraspecific trait variation to alter the outcome of the mutualism. In order for mean trait values to be useful as a functional trait to describe a species, as has been attempted for mycorrhizal response traits, interspecific variation must be much larger than intraspecific variation. While the variation among species has been extensively studied with respect to mycorrhizal response traits, variation within species has rarely been examined. We conducted a systematic review and analyzed how much variation for mycorrhizal growth and nutrient response typically exists within a plant species. We assessed 28 publications that included 60 individual studies testing mycorrhizal response in at least five genotypes of a plant species, and we found that intraspecific trait variation for mycorrhizal response was generally very large and highly variable depending on study design. The difference between the highest and lowest growth response in a study ranged from 10% to 350% across studies, and 36 of the studies included species for which both positive and negative growth responses to mycorrhizae were observed across different genotypes. The intraspecific variation for mycorrhizal growth response in some of these studies was larger than the variation documented among species across the plant kingdom. Phosphorus concentration and content was measured in 17 studies and variation in phosphorus response was similar to variation in growth responses. We also found that plant genotype was just as important for predicting mycorrhizal response as the effects of fungal inoculant identity. Our analysis highlights not only the potential importance of intraspecific trait variation for mycorrhizal response, but also the lack of research that has been done on the scale of this variation in plant species. Including intraspecific variation into research on the interactions between plants and their symbionts can increase our understanding of plant coexistence and ecological stability.

Broad diversity in monoterpene-sesquiterpene balance across wild sunflowers: Implications of leaf and floral volatiles for biotic interactions.

PREMISE: As plant lineages diversify across environmental gradients, species are predicted to encounter divergent biotic pressures. This study investigated the evolution of volatile secondary metabolism across species of Helianthus. METHODS: Leaves and petals of 40 species of wild Helianthus were analyzed via gas chromatography-mass spectrometry to determine volatile secondary metabolite profiles. RESULTS: Across all species, 500 compounds were identified; 40% were sesquiterpenes, 18% monoterpenes, 3% diterpenes, 4% fatty acid derivatives, and 35% other compounds such as phenolics and small organic molecules. Qualitatively, annuals and species from more arid western climates had leaf compositions with a higher proportion of total monoterpenes, while erect perennials and species from more mesic eastern habitats contained a higher proportion of total sesquiterpenes. Among species, mass-based leaf monoterpene and sesquiterpene abundance were identified as largely orthogonal axes of variation by principal component analysis. Profiles for leaves were not strongly correlated with those of petals. CONCLUSIONS: Volatile metabolites were highly diverse among wild Helianthus, indicating the value of this genus as a model system and rich genetic resource. The independence of leaf and petal volatile profiles indicates a low level of phenotypic integration between vegetative and reproductive structures, implying vegetative defense and reproductive defense or pollinator attraction functions mediated by terpene profiles in these two organs can evolve without major trade-offs. The major biosynthetic pathways for the major terpenes in wild Helianthus are already well described, providing a road map to deeper inquiry into the drivers of this diversity.

Genetic control of arbuscular mycorrhizal colonization by Rhizophagus intraradices in Helianthus annuus (L.).

Plant symbiosis with arbuscular mycorrhizal (AM) fungi provides many benefits, including increased nutrient uptake, drought tolerance, and belowground pathogen resistance. To develop a better understanding of the genetic architecture of mycorrhizal symbiosis, we conducted a genome-wide association study (GWAS) of this plant-fungal interaction in cultivated sunflower. A diversity panel of cultivated sunflower (Helianthus annuus L.) was phenotyped for root colonization under inoculation with the AM fungus Rhizophagus intraradices. Using a mixed linear model approach with a high-density genetic map, we identified genomic regions that are likely associated with R. intraradices colonization in sunflower. Additionally, we used a set of twelve diverse lines to assess the effect that inoculation with R. intraradices has on dried shoot biomass and macronutrient uptake. Colonization among lines in the mapping panel ranged from 0-70% and was not correlated with mycorrhizal growth response, shoot phosphorus response, or shoot potassium response among the Core 12 lines. Association mapping yielded three single-nucleotide polymorphisms (SNPs) that were significantly associated with R. intraradices colonization. This is the first study to use GWAS to identify genomic regions associated with AM colonization in an Asterid eudicot species. Three genes of interest identified from the regions containing these SNPs are likely related to plant defense.

Evolutionary trade-offs in the chemical defense of floral and fruit tissues across genus Cornus.

PREMISE: Defense investment in plant reproductive structures is relatively understudied compared to the defense of vegetative organs. Here the evolution of chemical defenses in reproductive structures is examined in light of the optimal defense, apparency, and resource availability hypotheses within the genus Cornus using a phylogenetic comparative approach in relation to phenology and native habitat environmental data. METHODS: Individuals representing 25 Cornus species were tracked for reproductive phenology over a full growing season at the Arnold Arboretum of Harvard University. Floral, fruit, and leaf tissue was sampled to quantify defensive chemistry as well as fruit nutritional traits relevant to bird dispersal. Native habitat environmental characteristics were estimated using locality data from digitized herbarium records coupled with global soil and climate data sets. RESULTS: The evolution of later flowering was correlated with increased floral tannins, and the evolution of later fruiting was correlated with increased total phenolics. Leaves were found to contain the highest tannin activity, while inflorescences contained the highest total flavonoids. Multiple aspects of fruit defensive chemistry were correlated with fruit nutritional traits. Floral and fruit defensive chemistry were evolutionarily correlated with aspects of native habitat temperature, precipitation, and soil characteristics. CONCLUSIONS: Results provide tentative support for the apparency hypothesis with respect to both flower and fruit phenology, while relative concentrations of secondary metabolites across organs provide mixed support for the optimal defense hypothesis. The evolution of reproductive defense with native habitat provides, at best, mixed support for the resource availability hypothesis.

Impact of genome duplication on secondary metabolite composition in non-cultivated species: a systematic meta-analysis.

BACKGROUND AND AIMS: Whole-genome duplication is known to influence ecological interactions and plant physiology; however, despite abundant case studies, much is still unknown about the typical impact of genome duplication on plant secondary metabolites (PSMs). In this study, we assessed the impact of polyploidy events on PSM characteristics in non-cultivated plants. METHODS: We conducted a systematic review and meta-analysis to compare composition and concentration of PSMs among closely related plant species or species complexes differing in ploidy level. KEY RESULTS: We assessed 53 studies that focus on PSMs among multiple cytotypes, of which only 14 studies compared concentration quantitatively among cytotypes. We found that whole-genome duplication can have a significant effect on PSM concentration; however, these effects are highly inconsistent. CONCLUSION: Overall, there was no consistent effect of whole-genome duplication on PSM concentrations or profiles.

Genome-Wide Association Mapping of Floral Traits in Cultivated Sunflower (Helianthus annuus).

Floral morphology and pigmentation are both charismatic and economically relevant traits associated with cultivated sunflower (Helianthus annuus L.). Recent work has linked floral morphology and pigmentation to pollinator efficiency and seed yield. Understanding the genetic architecture of such traits is essential for crop improvement, and gives insight into the role of genetic constraints in shaping floral diversity. A diversity panel of 288 sunflower genotypes was phenotyped for a variety of morphological, phenological, and color traits in both a greenhouse and a field setting. Association mapping was performed using 5788 SNP markers using a mixed linear model approach. Several dozen markers across 10 linkage groups were significantly associated with variation in morphological and color trait variation. Substantial trait plasticity was observed between greenhouse and field phenotyping, and associations differed between environments. Color traits mapped more strongly than morphology in both settings, with markers together explaining 16% of petal carotenoid content in the greenhouse, and 17% and 24% of variation in disc anthocyanin presence in the field and greenhouse, respectively. Morphological traits like disc size mapped more strongly in the field, with markers together explaining up to 19% of disc size variation. Loci identified here through association mapping within cultivated germplasm differ from those identified through biparental crosses between modern cultivated sunflower and either its wild progenitor or domesticated landraces. Several loci lie within genomic regions involved in domestication. Differences between phenotype expression under greenhouse and field conditions highlight the importance of plasticity in determining floral morphology and pigmentation.

Importance of whole-plant biomass allocation and reproductive timing to habitat differentiation across the North American sunflowers.

Background and Aims: Trait-based plant ecology attempts to use small numbers of functional traits to predict plant ecological strategies. However, a major gap exists between our understanding of organ-level ecophysiological traits and our understanding of whole-plant fitness and environmental adaptation. In this gap lie whole-plant organizational traits, including those that describe how plant biomass is allocated among organs and the timing of plant reproduction. This study explores the role of whole-plant organizational traits in adaptation to diverse environments in the context of life history, growth form and leaf economic strategy in a well-studied herbaceous system. Methods: A phylogenetic comparative approach was used in conjunction with common garden phenotyping to assess the evolution of biomass allocation and reproductive timing across 83 populations of 27 species of the diverse genus Helianthus (the sunflowers). Key Results: Broad diversity exists among species in both relative biomass allocation and reproductive timing. Early reproduction is strongly associated with resource-acquisitive leaf economic strategy, while biomass allocation is less integrated with either reproductive timing or leaf economics. Both biomass allocation and reproductive timing are strongly related to source site environmental characteristics, including length of the growing season, temperature, precipitation and soil fertility. Conclusions: Herbaceous taxa can adapt to diverse environments in many ways, including modulation of phenology, plant architecture and organ-level ecophysiology. Although leaf economic strategy captures one key aspect of plant physiology, on their own leaf traits are not particularly predictive of ecological strategies in Helianthus outside of the context of growth form, life history and whole-plant organization. These results highlight the importance of including data on whole-plant organization alongside organ-level ecophysiological traits when attempting to bridge the gap between functional traits and plant fitness and environmental adaptation.

Fine root tradeoffs between nitrogen concentration and xylem vessel traits preclude unified whole-plant resource strategies in Helianthus.

Recent work suggests variation in plant growth strategies is governed by a tradeoff in resource acquisition and use, ranging from a rapid resource acquisition strategy to a resource-conservative strategy. While evidence for this tradeoff has been found in leaves, knowledge of root trait strategies, and whether they reflect adaptive differentiation across environments, is limited. In the greenhouse, we investigated variation in fine root morphology (specific root length and tissue density), chemistry (nitrogen concentration and carbon:nitrogen), and anatomy (root cross-sectional traits) in populations of 26 Helianthus species and sister Phoebanthus tenuifolius. We also compared root trait variation in this study with leaf trait variation previously reported in a parallel study of these populations. Root traits varied widely and exhibited little phylogenetic signal, suggesting high evolutionary lability. Specific root length and root tissue density were weakly negatively correlated, but neither was associated with root nitrogen, providing little support for a single axis of root trait covariation. Correlations between traits measured in the greenhouse and native site characteristics were generally weak, suggesting a variety of equally viable root trait combinations exist within and across environments. However, high root nitrogen was associated with lower xylem vessel number and cross-sectional area, suggesting a tradeoff between nutrient investment and water transport capacity. This led to correlations between root and leaf traits that were not always consistent with an acquisition-conservation tradeoff at the whole-plant level. Given that roots must balance acquisition of water and nutrients with functions like anchorage, exudation, and microbial symbioses, the varied evidence for root trait covariation likely reflects the complexity of interacting selection pressures belowground. Similarly, the lack of evidence for a single acquisition-conservation tradeoff at the whole-plant level likely reflects the vastly different selection pressures shaping roots and leaves, and the resources they are optimized to obtain.

Macroevolution of leaf defenses and secondary metabolites across the genus Helianthus.

Leaf defenses are widely recognized as key adaptations and drivers of plant evolution. Across environmentally diverse habitats, the macroevolution of leaf defenses can be predicted by the univariate trade-off model, which predicts that defenses are functionally redundant and thus trade off, and the resource availability hypothesis, which predicts that defense investment is determined by inherent growth rate and that higher defense will evolve in lower resource environments. Here, we examined the evolution of leaf physical and chemical defenses and secondary metabolites in relation to environmental characteristics and leaf economic strategy across 28 species of Helianthus (the sunflowers). Using a phylogenetic comparative approach, we found few evolutionary trade-offs among defenses and no evidence for defense syndromes. We also found that leaf defenses are strongly related to leaf economic strategy, with higher defense in more resource-conservative species, although there is little support for the evolution of higher defense in low-resource habitats. A wide variety of physical and chemical defenses predict resistance to different insect herbivores, fungal pathogens, and a parasitic plant, suggesting that most sunflower defenses are not redundant in function and that wild Helianthus represents a rich source of variation for the improvement of crop sunflower.

Phylogenetic structural equation modelling reveals no need for an 'origin' of the leaf economics spectrum.

The leaf economics spectrum (LES) is a prominent ecophysiological paradigm that describes global variation in leaf physiology across plant ecological strategies using a handful of key traits. Nearly a decade ago, Shipley et al. (2006) used structural equation modelling to explore the causal functional relationships among LES traits that give rise to their strong global covariation. They concluded that an unmeasured trait drives LES covariation, sparking efforts to identify the latent physiological trait underlying the 'origin' of the LES. Here, we use newly developed phylogenetic structural equation modelling approaches to reassess these conclusions using both global LES data as well as data collected across scales in the genus Helianthus. For global LES data, accounting for phylogenetic non-independence indicates that no additional unmeasured traits are required to explain LES covariation. Across datasets in Helianthus, trait relationships are highly variable, indicating that global-scale models may poorly describe LES covariation at non-global scales.

Evolution of the leaf economics spectrum in herbs: Evidence from environmental divergences in leaf physiology across Helianthus (Asteraceae).

The leaf economics spectrum (LES) describes a major axis of plant functional trait variation worldwide, defining suites of leaf traits aligned with resource-acquisitive to resource-conservative ecological strategies. The LES has been interpreted to arise from leaf-level trade-offs among ecophysiological traits common to all plants. However, it has been suggested that the defining leaf-level trade-offs of the LES may not hold within specific functional groups (e.g., herbs) nor within many groups of closely related species, which challenges the usefulness of the LES paradigm across evolutionary scales. Here, we examine the evolution of the LES across 28 species of the diverse herbaceous genus Helianthus (the sunflowers), which occupies a wide range of habitats and climate variation across North America. Using a phylogenetic comparative approach, we find repeated evolution of more resource-acquisitive LES strategies in cooler, drier, and more fertile environments. We also find macroevolutionary correlations among LES traits that recapitulate aspects of the global LES, but with one major difference: leaf mass per area is uncorrelated with leaf lifespan. This indicates that whole-plant processes likely drive variation in leaf lifespan across Helianthus, rather than leaf-level trade-offs. These results suggest that LES patterns do not reflect universal physiological trade-offs at small evolutionary scales.

Species tree estimation of diploid Helianthus (Asteraceae) using target enrichment.

PREMISE OF THE STUDY: The sunflower genus Helianthus has long been recognized as economically significant, containing species of agricultural and horticultural importance. Additionally, this genus displays a large range of phenotypic and genetic variation, making Helianthus a useful system for studying evolutionary and ecological processes. Here we present the most robust Helianthus phylogeny to date, laying the foundation for future studies of this genus. METHODS: We used a target enrichment approach across 37 diploid Helianthus species/subspecies with a total of 103 accessions. This technique garnered 170 genes used for both coalescent and concatenation analyses. The resulting phylogeny was additionally used to examine the evolution of life history and growth form across the genus. KEY RESULTS: Coalescent and concatenation approaches were largely congruent, resolving a large annual clade and two large perennial clades. However, several relationships deeper within the phylogeny were more weakly supported and incongruent among analyses including the placement of H. agrestis, H. cusickii, H. gracilentus, H. mollis, and H. occidentalis. CONCLUSIONS: The current phylogeny supports three major clades including a large annual clade, a southeastern perennial clade, and another clade of primarily large-statured perennials. Relationships among taxa are more consistent with early phylogenies of the genus using morphological and crossing data than recent efforts using single genes, which highlight the difficulties of phylogenetic estimation in genera known for reticulate evolution. Additionally, conflict and low support at the base of the perennial clades may suggest a rapid radiation and/or ancient introgression within the genus.

Does investment in leaf defenses drive changes in leaf economic strategy? A focus on whole-plant ontogeny.

Leaf defenses have long been studied in the context of plant growth rate, resource availability, and optimal investment theory. Likewise, one of the central modern paradigms of plant ecophysiology, the leaf economics spectrum (LES), has been extensively studied in the context of these factors across ecological scales ranging from global species data sets to temporal shifts within individuals. Despite strong physiological links between LES strategy and leaf defenses in structure, function, and resource investment, the relationship between these trait classes has not been well explored. This study investigates the relationship between leaf defenses and LES strategy across whole-plant ontogeny in three diverse Helianthus species known to exhibit dramatic ontogenetic shifts in LES strategy, focusing primarily on physical and quantitative chemical defenses. Plants were grown under controlled environmental conditions and sampled for LES and defense traits at four ontogenetic stages. Defenses were found to shift strongly with ontogeny, and to correlate strongly with LES strategy. More advanced ontogenetic stages with more conservative LES strategy leaves had higher tannin activity and toughness in all species, and higher leaf dry matter content in two of three species. Modeling results in two species support the conclusion that changes in defenses drive changes in LES strategy through ontogeny, and in one species that changes in defenses and LES strategy are likely independently driven by ontogeny. Results of this study support the hypothesis that leaf-level allocation to defenses might be an important determinant of leaf economic traits, where high investment in defenses drives a conservative LES strategy.

Leaf mass per area is independent of vein length per area: avoiding pitfalls when modelling phenotypic integration (reply to Blonder et al. 2014).

It has been recently proposed that leaf vein length per area (VLA) is the major determinant of leaf mass per area ( MA), and would thereby determine other traits of the leaf economic spectrum (LES), such as photosynthetic rate per mass (A(mass)), nitrogen concentration per mass (N(mass)) and leaf lifespan (LL). In a previous paper we argued that this 'vein origin' hypothesis was supported only by a mathematical model with predestined outcomes, and that we found no support for the 'vein origin' hypothesis in our analyses of compiled data. In contrast to the 'vein origin' hypothesis, empirical evidence indicated that VLA and LMA are independent mechanistically, and VLA (among other vein traits) contributes to a higher photosynthetic rate per area (A(area)), which scales up to driving a higher A(mass), all independently of LMA, N(mass) and LL. In their reply to our paper, Blonder et al. (2014) raised questions about our analysis of their model, but did not address our main point, that the data did not support their hypothesis. In this paper we provide further analysis of an extended data set, which again robustly demonstrates the mechanistic independence of LMA from VLA, and thus does not support the 'vein origin' hypothesis. We also address the four specific points raised by Blonder et al. (2014) regarding our analyses. We additionally show how this debate provides critical guidance for improved modelling of LES traits and other networks of phenotypic traits that determine plant performance under contrasting environments.

Adaptive differentiation of traits related to resource use in a desert annual along a resource gradient.

• Plant resource-use traits are generally hypothesized to be adaptively differentiated for populations distributed along resource gradients. Although nutrient limitations are expected to select for resource-conservative strategies, water limitations may select for either resource-conservative or -acquisitive strategies. We test whether population differentiation reflects local adaptation for traits associated with resource-use strategies in a desert annual (Helianthus anomalus) distributed along a gradient of positively covarying water and nutrient availability. • We compared quantitative trait variation (Q(ST)) with neutral genetic differentiation (F(ST)), in a common garden glasshouse study, for leaf economics spectrum (LES) and related traits: photosynthesis (A(mass), A(area)), leaf nitrogen (N(mass), N(area)), leaf lifetime (LL), leaf mass per area (LMA), leaf water content (LWC), water-use efficiency (WUE, estimated as δ(13)C) and days to first flower (DFF). • Q(ST)-F(ST) differences support adaptive differentiation for Amass , N(mass), N(area), LWC and DFF. The trait combinations associated with drier and lower fertility sites represent correlated trait evolution consistent with the more resource-acquisitive end of the LES. There was no evidence for adaptive differentiation for A(area), LMA and WUE. • These results demonstrate that hot dry environments can selectively favor correlated evolution of traits contributing to a resource-acquisitive and earlier reproduction 'escape' strategy, despite lower fertility.

How do leaf veins influence the worldwide leaf economic spectrum? Review and synthesis.

Leaf vein traits are implicated in the determination of gas exchange rates and plant performance. These traits are increasingly considered as causal factors affecting the 'leaf economic spectrum' (LES), which includes the light-saturated rate of photosynthesis, dark respiration, foliar nitrogen concentration, leaf dry mass per area (LMA) and leaf longevity. This article reviews the support for two contrasting hypotheses regarding a key vein trait, vein length per unit leaf area (VLA). Recently, Blonder et al. (2011, 2013) proposed that vein traits, including VLA, can be described as the 'origin' of the LES by structurally determining LMA and leaf thickness, and thereby vein traits would predict LES traits according to specific equations. Careful re-examination of leaf anatomy, published datasets, and a newly compiled global database for diverse species did not support the 'vein origin' hypothesis, and moreover showed that the apparent power of those equations to predict LES traits arose from circularity. This review provides a 'flux trait network' hypothesis for the effects of vein traits on the LES and on plant performance, based on a synthesis of the previous literature. According to this hypothesis, VLA, while virtually independent of LMA, strongly influences hydraulic conductance, and thus stomatal conductance and photosynthetic rate. We also review (i) the specific physiological roles of VLA; (ii) the role of leaf major veins in influencing LES traits; and (iii) the role of VLA in determining photosynthetic rate per leaf dry mass and plant relative growth rate. A clear understanding of leaf vein traits provides a new perspective on plant function independently of the LES and can enhance the ability to explain and predict whole plant performance under dynamic conditions, with applications towards breeding improved crop varieties.

Ontogeny strongly and differentially alters leaf economic and other key traits in three diverse Helianthus species.

The leaf economics spectrum (LES) describes large cross-species variation in suites of leaf functional traits ranging from resource-acquisitive to resource-conservative strategies. Such strategies have been integral in explaining plant adaptation to diverse environments, and have been linked to numerous ecosystem processes. The LES has previously been found to be significantly modulated by climate, soil fertility, biogeography, growth form, and life history. One largely unexplored aspect of LES variation, whole-plant ontogeny, is investigated here using multiple populations of three very different species of sunflower: Helianthus annuus, Helianthus mollis, and Helianthus radula. Plants were grown under environmentally controlled conditions and assessed for LES and related traits at four key developmental stages, using recently matured leaves to standardize for leaf age. Nearly every trait exhibited a significant ontogenetic shift in one or more species, with trait patterns differing among populations and species. Photosynthetic rate, leaf nitrogen concentration, and leaf mass per area exhibited surprisingly large changes, spanning over two-thirds of the original cross-species LES variation and shifting from resource-acquisitive to resource-conservative strategies as the plants matured. Other traits being investigated in relation to the LES, such as leaf water content, pH, and vein density, also showed large changes. The finding that ontogenetic variation in LES strategy can be substantial leads to a recommendation of standardization by developmental stage when assessing 'species values' of labile traits for comparative approaches. Additionally, the substantial ontogenetic trait shifts seen within single individuals provide an opportunity to uncover the contribution of gene regulatory changes to variation in LES traits.