Massive haplotypes underlie ecotypic differentiation in sunflowers.

Species often include multiple ecotypes that are adapted to different environments1. However, it is unclear how ecotypes arise and how their distinctive combinations of adaptive alleles are maintained despite hybridization with non-adapted populations2-4. Here, by resequencing 1,506 wild sunflowers from 3 species (Helianthus annuus, Helianthus petiolaris and Helianthus argophyllus), we identify 37 large (1-100 Mbp in size), non-recombining haplotype blocks that are associated with numerous ecologically relevant traits, as well as soil and climate characteristics. Limited recombination in these haplotype blocks keeps adaptive alleles together, and these regions differentiate sunflower ecotypes. For example, haplotype blocks control a 77-day difference in flowering between ecotypes of the silverleaf sunflower H. argophyllus (probably through deletion of a homologue of FLOWERING LOCUS T (FT)), and are associated with seed size, flowering time and soil fertility in dune-adapted sunflowers. These haplotypes are highly divergent, frequently associated with structural variants and often appear to represent introgressions from other-possibly now-extinct-congeners. These results highlight a pervasive role of structural variation in ecotypic adaptation.

The genomic basis of nitrogen utilization efficiency and trait plasticity to improve nutrient stress tolerance in cultivated sunflower.

Maintaining crop productivity is challenging as population growth, climate change, and increasing fertilizer costs necessitate expanding crop production to poorer lands whilst reducing inputs. Enhancing crops' nutrient use efficiency is thus an important goal, but requires a better understanding of related traits and their genetic basis. We investigated variation in low nutrient stress tolerance in a diverse panel of cultivated sunflower genotypes grown under high and low nutrient conditions, assessing relative growth rate (RGR) as performance. We assessed variation in traits related to nitrogen utilization efficiency (NUtE), mass allocation, and leaf elemental content. Across genotypes, nutrient limitation generally reduced RGR. Moreover, there was a negative correlation between vigor (RGR in control) and decline in RGR in response to stress. Given this trade-off, we focused on nutrient stress tolerance independent of vigor. This tolerance metric correlated with the change in NUtE, plasticity for a suite of morphological traits, and leaf element content. Genome-wide associations revealed regions associated with variation and plasticity in multiple traits, including two regions with seemingly additive effects on NUtE change. Our results demonstrate potential avenues for improving sunflower nutrient stress tolerance independent of vigor, and highlight specific traits and genomic regions that could play a role in enhancing tolerance.

Similar Transcriptomic Responses to Early and Late Drought Stresses Produce Divergent Phenotypes in Sunflower (Helianthus annuus L.).

Cultivated sunflower (Helianthus annuus L.) exhibits numerous phenotypic and transcriptomic responses to drought. However, the ways in which these responses vary with differences in drought timing and severity are insufficiently understood. We used phenotypic and transcriptomic data to evaluate the response of sunflower to drought scenarios of different timing and severity in a common garden experiment. Using a semi-automated outdoor high-throughput phenotyping platform, we grew six oilseed sunflower lines under control and drought conditions. Our results reveal that similar transcriptomic responses can have disparate phenotypic effects when triggered at different developmental time points. Leaf transcriptomic responses, however, share similarities despite timing and severity differences (e.g., 523 differentially expressed genes (DEGs) were shared across all treatments), though increased severity elicited greater differences in expression, particularly during vegetative growth. Across treatments, DEGs were highly enriched for genes related to photosynthesis and plastid maintenance. A co-expression analysis identified a single module (M8) enriched in all drought stress treatments. Genes related to drought, temperature, proline biosynthesis, and other stress responses were overrepresented in this module. In contrast to transcriptomic responses, phenotypic responses were largely divergent between early and late drought. Early-stressed sunflowers responded to drought with reduced overall growth, but became highly water-acquisitive during recovery irrigation, resulting in overcompensation (higher aboveground biomass and leaf area) and a greater overall shift in phenotypic correlations, whereas late-stressed sunflowers were smaller and more water use-efficient. Taken together, these results suggest that drought stress at an earlier growth stage elicits a change in development that enables greater uptake and transpiration of water during recovery, resulting in higher growth rates despite similar initial transcriptomic responses.

Key Traits and Genes Associate with Salinity Tolerance Independent from Vigor in Cultivated Sunflower.

With rising food demands, crop production on salinized lands is increasingly necessary. Sunflower (Helianthus annuus), a moderately salt-tolerant crop, exhibits a tradeoff where more vigorous, high-performing genotypes have a greater proportional decline in biomass under salinity stress. Prior research has found deviations from this relationship across genotypes. Here, we identified the traits and genomic regions underlying variation in this expectation-deviation tolerance (the magnitude and direction of deviations from the expected effect of salinity). We grew a sunflower diversity panel under control and salt-stressed conditions and measured a suite of morphological (growth, mass allocation, plant and leaf morphology) and leaf ionomic traits. The genetic basis of variation and plasticity in these traits was investigated via genome-wide association, which also enabled the identification of genomic regions (i.e. haplotypic blocks) influencing multiple traits. We found that the magnitude and direction of plasticity in whole-root mass fraction, fine root mass fraction, and chlorophyll content, as well as leaf sodium and potassium content under saline conditions, were most strongly correlated with expectation-deviation tolerance. We identified multiple genomic regions underlying these traits as well as a single alpha-mannosidase gene directly associated with this tolerance metric. Our results show that, by taking the vigor-salinity effect tradeoff into account, we can identify unique traits and genes associated with salinity tolerance. Since these traits and genomic regions are distinct from those associated with high vigor (i.e. growth in benign conditions), they provide an avenue for increasing salinity tolerance in high-performing sunflower genotypes without compromising vigor.

Revised Phylogeny of the Cellulose Synthase Gene Superfamily: Insights into Cell Wall Evolution.

Cell walls are crucial for the integrity and function of all land plants and are of central importance in human health, livestock production, and as a source of renewable bioenergy. Many enzymes that mediate the biosynthesis of cell wall polysaccharides are encoded by members of the large cellulose synthase (CesA) gene superfamily. Here, we analyzed 29 sequenced genomes and 17 transcriptomes to revise the phylogeny of the CesA gene superfamily in angiosperms. Our results identify ancestral gene clusters that predate the monocot-eudicot divergence and reveal several novel evolutionary observations, including the expansion of the Poaceae-specific cellulose synthase-like CslF family to the graminids and restiids and the characterization of a previously unreported eudicot lineage, CslM, that forms a reciprocally monophyletic eudicot-monocot grouping with the CslJ clade. The CslM lineage is widely distributed in eudicots, and the CslJ clade, which was thought previously to be restricted to the Poales, is widely distributed in monocots. Our analyses show that some members of the CslJ lineage, but not the newly identified CslM genes, are capable of directing (1,3;1,4)-ß-glucan biosynthesis, which, contrary to current dogma, is not restricted to Poaceae.

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.

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.

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.

Plant hydraulics as a central hub integrating plant and ecosystem function: meeting report for 'Emerging Frontiers in Plant Hydraulics' (Washington, DC, May 2015).

Water plays a central role in plant biology and the efficiency of water transport throughout the plant affects both photosynthetic rate and growth, an influence that scales up deterministically to the productivity of terrestrial ecosystems. Moreover, hydraulic traits mediate the ways in which plants interact with their abiotic and biotic environment. At landscape to global scale, plant hydraulic traits are important in describing the function of ecological communities and ecosystems. Plant hydraulics is increasingly recognized as a central hub within a network by which plant biology is connected to palaeobiology, agronomy, climatology, forestry, community and ecosystem ecology and earth-system science. Such grand challenges as anticipating and mitigating the impacts of climate change, and improving the security and sustainability of our food supply rely on our fundamental knowledge of how water behaves in the cells, tissues, organs, bodies and diverse communities of plants. A workshop, 'Emerging Frontiers in Plant Hydraulics' supported by the National Science Foundation, was held in Washington DC, 2015 to promote open discussion of new ideas, controversies regarding measurements and analyses, and especially, the potential for expansion of up-scaled and down-scaled inter-disciplinary research, and the strengthening of connections between plant hydraulic research, allied fields and global modelling efforts.

Evidence of correlated evolution and adaptive differentiation of stem and leaf functional traits in the herbaceous genus, Helianthus.

PREMISE OF THE STUDY: Patterns of plant stem traits are expected to align with a "fast-slow" plant economic spectrum across taxa. Although broad patterns support such tradeoffs in field studies, tests of hypothesized correlated trait evolution and adaptive differentiation are more robust when taxa relatedness and environment are taken into consideration. Here we test for correlated evolution of stem and leaf traits and their adaptive differentiation across environments in the herbaceous genus, Helianthus. METHODS: Stem and leaf traits of 14 species of Helianthus (28 populations) were assessed in a common garden greenhouse study. Phylogenetically independent contrasts were used to test for evidence of correlated evolution of stem hydraulic and biomechanical properties, correlated evolution of stem and leaf traits, and adaptive differentiation associated with source habitat environments. KEY RESULTS: Among stem traits, there was evidence for correlated evolution of some hydraulic and biomechanical properties, supporting an expected tradeoff between stem theoretical hydraulic efficiency and resistance to bending stress. Population differentiation for suites of stem and leaf traits was found to be consistent with a "fast-slow" resource-use axis for traits related to water transport and use. Associations of population traits with source habitat characteristics supported repeated evolution of a resource-acquisitive "drought-escape" strategy in arid environments. CONCLUSIONS: This study provides evidence of correlated evolution of stem and leaf traits consistent with the fast-slow spectrum of trait combinations related to water transport and use along the stem-to-leaf pathway. Correlations of traits with source habitat characteristics further indicate that the correlated evolution is associated, at least in part, with adaptive differentiation of Helianthus populations among native habitats differing in climate.

Genetics and physiology of cell wall polysaccharides in the model C4 grass, Setaria viridis spp.

BACKGROUND: Setaria viridis has emerged as a model species for the larger C4 grasses. Here the cellulose synthase (CesA) superfamily has been defined, with an emphasis on the amounts and distribution of (1,3;1,4)-ß-glucan, a cell wall polysaccharide that is characteristic of the grasses and is of considerable value for human health. METHODS: Orthologous relationship of the CesA and Poales-specific cellulose synthase-like (Csl) genes among Setaria italica (Si), Sorghum bicolor (Sb), Oryza sativa (Os), Brachypodium distachyon (Bradi) and Hordeum vulgare (Hv) were compared using bioinformatics analysis. Transcription profiling of Csl gene families, which are involved in (1,3;1,4)-ß-glucan synthesis, was performed using real-time quantitative PCR (Q-PCR). The amount of (1,3;1,4)-ß-glucan was measured using a modified Megazyme assay. The fine structures of the (1,3;1,4)-ß-glucan, as denoted by the ratio of cellotriosyl to cellotetraosyl residues (DP3:DP4 ratio) was assessed by chromatography (HPLC and HPAEC-PAD). The distribution and deposition of the MLG was examined using the specific antibody BG-1 and captured using fluorescence and transmission electron microscopy (TEM). RESULTS: The cellulose synthase gene superfamily contains 13 CesA and 35 Csl genes in Setaria. Transcript profiling of CslF, CslH and CslJ gene families across a vegetative tissue series indicated that SvCslF6 transcripts were the most abundant relative to all other Csl transcripts. The amounts of (1,3;1,4)-ß-glucan in Setaria vegetative tissues ranged from 0.2% to 2.9% w/w with much smaller amounts in developing grain (0.003% to 0.013% w/w). In general, the amount of (1,3;1,4)-ß-glucan was greater in younger than in older tissues. The DP3:DP4 ratios varied between tissue types and across developmental stages, and ranged from 2.4 to 3.0:1. The DP3:DP4 ratios in developing grain ranged from 2.5 to 2.8:1. Micrographs revealing the distribution of (1,3;1,4)-ß-glucan in walls of different cell types and the data were consistent with the quantitative (1,3;1,4)-ß-glucan assays. CONCLUSION: The characteristics of the cellulose synthase gene superfamily and the accumulation and distribution of (1,3;1,4)-ß-glucans in Setaria are similar to those in other C4 grasses, including sorghum. This suggests that Setaria is a suitable model plant for cell wall polysaccharide biology in C4 grasses.

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.

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.

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.

Cultivated sunflower (Helianthus annuus L.) has lower tolerance of moderate drought stress than its con-specific wild relative, but the underlying traits remain elusive.

Cultivated crops are generally expected to have less abiotic stress tolerance than their wild relatives. However, this assumption is not well supported by empirical literature and may depend on the type of stress and how it is imposed, as well as the measure of tolerance being used. Here, we investigated whether wild and cultivated accessions of Helianthus annuus differed in stress tolerance assessed as proportional decline in biomass due to drought and whether wild and cultivated accessions differed in trait responses to drought and trait associations with tolerance. In a greenhouse study, H. annuus accessions in the two domestication classes (eight cultivated and eight wild accessions) received two treatments: a well-watered control and a moderate drought implemented as a dry down followed by maintenance at a predetermined soil moisture level with automated irrigation. Treatments were imposed at the seedling stage, and plants were harvested after 2 weeks of treatment. The proportional biomass decline in response to drought was 24% for cultivated H. annuus accessions but was not significant for the wild accessions. Thus, using the metric of proportional biomass decline, the cultivated accessions had less drought tolerance. Among accessions, there was no tradeoff between drought tolerance and vigor assessed as biomass in the control treatment. In a multivariate analysis, wild and cultivated accessions did not differ from each other or in response to drought for a subset of morphological, physiological, and allocational traits. Analyzed individually, traits varied in response to drought in wild and/or cultivated accessions, including declines in specific leaf area, leaf theoretical maximum stomatal conductance (gsmax), and stomatal pore length, but there was no treatment response for stomatal density, succulence, or the ability to osmotically adjust. Focusing on traits associations with tolerance, plasticity in gsmax was the most interesting because its association with tolerance differed by domestication class (although the effects were relatively weak) and thus might contribute to lower tolerance of cultivated sunflower. Our H. annuus results support the expectation that stress tolerance is lower in crops than wild relatives under some conditions. However, determining the key traits that underpin differences in moderate drought tolerance between wild and cultivated H. annuus remains elusive.

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.

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.

High genetic diversity and low population structure in Porter's sunflower (Helianthus porteri).

Granite outcrops in the southeastern United States are rare and isolated habitats that support edaphically controlled communities dominated by herbaceous plants. They harbor rare and endemic species that are expected to have low genetic variability and high population structure due to small population sizes and their disjunct habitat. We test this expectation for an annual outcrop endemic, Helianthus porteri (Porter's sunflower). Contrary to expectation, H. porteri has relatively high genetic diversity (H e = 0.681) and relatively low genetic structure among the native populations (F ST = 0.077) when compared to 5 other Helianthus species (N = 288; 18 expressed sequence tag-SSR markers). These findings suggest greater gene flow than expected. The potential for gene flow is supported by the analysis of transplant populations established with propagules from a common source in 1959. One population established close to a native population (1.5 km) at the edge of the natural range is genetically similar to and shares rare alleles with the adjacent native population and is distinct from the central source population. In contrast, a transplant population established north of the native range has remained similar to the source population. The relatively high genetic diversity and low population structure of this species, combined with the long-term success of transplanted populations, bode well for its persistence as long as the habitat persists.