Exome resequencing and GWAS for growth, ecophysiology, and chemical and metabolomic composition of wood of Populus trichocarpa.

BACKGROUND: Populus trichocarpa is an important forest tree species for the generation of lignocellulosic ethanol. Understanding the genomic basis of biomass production and chemical composition of wood is fundamental in supporting genetic improvement programs. Considerable variation has been observed in this species for complex traits related to growth, phenology, ecophysiology and wood chemistry. Those traits are influenced by both polygenic control and environmental effects, and their genome architecture and regulation are only partially understood. Genome wide association studies (GWAS) represent an approach to advance that aim using thousands of single nucleotide polymorphisms (SNPs). Genotyping using exome capture methodologies represent an efficient approach to identify specific functional regions of genomes underlying phenotypic variation. RESULTS: We identified 813 K SNPs, which were utilized for genotyping 461 P. trichocarpa clones, representing 101 provenances collected from Oregon and Washington, and established in California. A GWAS performed on 20 traits, considering single SNP-marker tests identified a variable number of significant SNPs (p-value < 6.1479E-8) in association with diameter, height, leaf carbon and nitrogen contents, and δ15N. The number of significant SNPs ranged from 2 to 220 per trait. Additionally, multiple-marker analyses by sliding-windows tests detected between 6 and 192 significant windows for the analyzed traits. The significant SNPs resided within genes that encode proteins belonging to different functional classes as such protein synthesis, energy/metabolism and DNA/RNA metabolism, among others. CONCLUSIONS: SNP-markers within genes associated with traits of importance for biomass production were detected. They contribute to characterize the genomic architecture of P. trichocarpa biomass required to support the development and application of marker breeding technologies.

Exploiting Differential Gene Expression and Epistasis to Discover Candidate Genes for Drought-Associated QTLs in Arabidopsis thaliana.

Soil water availability represents one of the most important selective agents for plants in nature and the single greatest abiotic determinant of agricultural productivity, yet the genetic bases of drought acclimation responses remain poorly understood. Here, we developed a systems-genetic approach to characterize quantitative trait loci (QTLs), physiological traits and genes that affect responses to soil moisture deficit in the TSUxKAS mapping population of Arabidopsis thaliana. To determine the effects of candidate genes underlying QTLs, we analyzed gene expression as a covariate within the QTL model in an effort to mechanistically link markers, RNA expression, and the phenotype. This strategy produced ranked lists of candidate genes for several drought-associated traits, including water use efficiency, growth, abscisic acid concentration (ABA), and proline concentration. As a proof of concept, we recovered known causal loci for several QTLs. For other traits, including ABA, we identified novel loci not previously associated with drought. Furthermore, we documented natural variation at two key steps in proline metabolism and demonstrated that the mitochondrial genome differentially affects genomic QTLs to influence proline accumulation. These findings demonstrate that linking genome, transcriptome, and phenotype data holds great promise to extend the utility of genetic mapping, even when QTL effects are modest or complex.

Variation in MPK12 affects water use efficiency in Arabidopsis and reveals a pleiotropic link between guard cell size and ABA response.

Plant water relations are critical for determining the distribution, persistence, and fitness of plant species. Studying the genetic basis of ecologically relevant traits, however, can be complicated by their complex genetic, physiological, and developmental basis and their interaction with the environment. Water use efficiency (WUE), the ratio of photosynthetic carbon assimilation to stomatal conductance to water, is a dynamic trait with tremendous ecological and agricultural importance whose genetic control is poorly understood. In the present study, we use a quantitative trait locus-mapping approach to locate, fine-map, clone, confirm, and characterize an allelic substitution that drives differences in WUE among natural accessions of Arabidopsis thaliana. We show that a single amino acid substitution in an abscisic acid-responsive kinase, AtMPK12, causes reduction in WUE, and we confirm its functional role using transgenics. We further demonstrate that natural alleles at AtMPK12 differ in their response to cellular and environmental cues, with the allele from the Cape Verde Islands (CVI) being less responsive to hormonal inhibition of stomatal opening and more responsive to short-term changes in vapor pressure deficit. We also show that the CVI allele results in constitutively larger stomata. Together, these differences cause higher stomatal conductance and lower WUE compared with the common allele. These physiological changes resulted in reduced whole-plant transpiration efficiency and reduced fitness under water-limited compared with well-watered conditions. Our work demonstrates how detailed analysis of naturally segregating functional variation can uncover the molecular and physiological basis of a key trait associated with plant performance in ecological and agricultural settings.

Expression quantitative trait locus mapping across water availability environments reveals contrasting associations with genomic features in Arabidopsis.

The regulation of gene expression is crucial for an organism's development and response to stress, and an understanding of the evolution of gene expression is of fundamental importance to basic and applied biology. To improve this understanding, we conducted expression quantitative trait locus (eQTL) mapping in the Tsu-1 (Tsushima, Japan) × Kas-1 (Kashmir, India) recombinant inbred line population of Arabidopsis thaliana across soil drying treatments. We then used genome resequencing data to evaluate whether genomic features (promoter polymorphism, recombination rate, gene length, and gene density) are associated with genes responding to the environment (E) or with genes with genetic variation (G) in gene expression in the form of eQTLs. We identified thousands of genes that responded to soil drying and hundreds of main-effect eQTLs. However, we identified very few statistically significant eQTLs that interacted with the soil drying treatment (GxE eQTL). Analysis of genome resequencing data revealed associations of several genomic features with G and E genes. In general, E genes had lower promoter diversity and local recombination rates. By contrast, genes with eQTLs (G) had significantly greater promoter diversity and were located in genomic regions with higher recombination. These results suggest that genomic architecture may play an important a role in the evolution of gene expression.

Natural variation in abiotic stress responsive gene expression and local adaptation to climate in Arabidopsis thaliana.

Gene expression varies widely in natural populations, yet the proximate and ultimate causes of this variation are poorly known. Understanding how variation in gene expression affects abiotic stress tolerance, fitness, and adaptation is central to the field of evolutionary genetics. We tested the hypothesis that genes with natural genetic variation in their expression responses to abiotic stress are likely to be involved in local adaptation to climate in Arabidopsis thaliana. Specifically, we compared genes with consistent expression responses to environmental stress (expression stress responsive, "eSR") to genes with genetically variable responses to abiotic stress (expression genotype-by-environment interaction, "eGEI"). We found that on average genes that exhibited eGEI in response to drought or cold had greater polymorphism in promoter regions and stronger associations with climate than those of eSR genes or genomic controls. We also found that transcription factor binding sites known to respond to environmental stressors, especially abscisic acid responsive elements, showed significantly higher polymorphism in drought eGEI genes in comparison to eSR genes. By contrast, eSR genes tended to exhibit relatively greater pairwise haplotype sharing, lower promoter diversity, and fewer nonsynonymous polymorphisms, suggesting purifying selection or selective sweeps. Our results indicate that cis-regulatory evolution and genetic variation in stress responsive gene expression may be important mechanisms of local adaptation to climatic selective gradients.

Physiological genomics of response to soil drying in diverse Arabidopsis accessions.

Arabidopsis thaliana, like many species, is characterized by abundant genetic variation. This variation is rapidly being cataloged at the sequence level, but careful dissection of genetic variation in whole-organism responses to stresses encountered in the natural environment are lacking; this functional variation can be exploited as a natural mutant screen to determine gene function. Here, we document physiological and transcriptomic response to soil drying in 17 natural accessions of Arabidopsis. By imposing ecologically realistic stress conditions, we found that acclimation in Arabidopsis involved a strong signature of increased investment in photosynthesis, carbohydrate turnover, and root growth. Our results extend previous work in the Columbia accession suggesting that abscisic acid signaling pathways play an important role in drought stress response. We also identified several mechanisms, including an increase in leaf nitrogen concentration and upregulation of two-component signaling relays, that were common to most natural accessions but had not been identified in studies using only the Columbia accession. Principal component analysis reveals strong correlations between suites of genes and specific physiological responses to stress. The functional variants we identified may represent adaptive mutations in natural habitats and useful variants for agronomic improvement of crop species.

The physiological basis for genetic variation in water use efficiency and carbon isotope composition in Arabidopsis thaliana.

Ecologists and physiologists have documented extensive variation in water use efficiency (WUE) in Arabidopsis thaliana, as well as association of WUE with climatic variation. Here, we demonstrate correlations of whole-plant transpiration efficiency and carbon isotope composition (δ(13)C) among life history classes of A. thaliana. We also use a whole-plant cuvette to examine patterns of co-variation in component traits of WUE and δ(13)C. We find that stomatal conductance (g s) explains more variation in WUE than does A. Overall, there was a strong genetic correlation between A and g s, consistent with selection acting on the ratio of these traits. At a more detailed level, genetic variation in A was due to underlying variation in both maximal rate of carboxylation (V cmax) and maximum electron transport rate (Jmax). We also found strong effects of leaf anatomy, where lines with lower WUE had higher leaf water content (LWC) and specific leaf area (SLA), suggesting a role for mesophyll conductance (g m) in variation of WUE. We hypothesize that this is due to an effect through g m, and test this hypothesis using the abi4 mutant. We show that mutants of ABI4 have higher SLA, LWC, and g m than wild-type, consistent with variation in leaf anatomy causing variation in g m and δ(13)C. These functional data also add further support to the central, integrative role of ABI4 in simultaneously altering ABA sensitivity, sugar signaling, and CO2 assimilation. Together our results highlight the need for a more holistic approach in functional studies, both for more accurate annotation of gene function and to understand co-limitations to plant growth and productivity.

Pre-dawn stomatal opening does not substantially enhance early-morning photosynthesis in Helianthus annuus.

Most C3 plant species have partially open stomata during the night especially in the 3-5 h before dawn. This pre-dawn stomatal opening has been hypothesized to enhance early-morning photosynthesis (A) by reducing diffusion limitations to CO2 at dawn. We tested this hypothesis in cultivated Helianthus annuus using whole-shoot gas exchange, leaf level gas exchange and modelling approaches. One hour pre-dawn low-humidity treatments were used to reduce pre-dawn stomatal conductance (g). At the whole-shoot level, a difference of pre-dawn g (0.40 versus 0.17 mol m(-2) s(-1)) did not significantly affect A during the first hour after dawn. Shorter term effects were investigated with leaf level gas exchange measurements and a difference of pre-dawn g (0.10 versus 0.04 mol m(-2) s(-1)) affected g and A for only 5 min after dawn. The potential effects of a wider range of stomatal apertures were explored with an empirical model of the relationship between A and intercellular CO2 concentration during the half-hour after dawn. Modelling results demonstrated that even extremely low pre-dawn stomatal conductance values have only a minimal effect on early-morning A for a few minutes after dawn. Thus, we found no evidence that pre-dawn stomatal opening enhances A.

Pleiotropy of FRIGIDA enhances the potential for multivariate adaptation.

An evolutionary response to selection requires genetic variation; however, even if it exists, then the genetic details of the variation can constrain adaptation. In the simplest case, unlinked loci and uncorrelated phenotypes respond directly to multivariate selection and permit unrestricted paths to adaptive peaks. By contrast, 'antagonistic' pleiotropic loci may constrain adaptation by affecting variation of many traits and limiting the direction of trait correlations to vectors that are not favoured by selection. However, certain pleiotropic configurations may improve the conditions for adaptive evolution. Here, we present evidence that the Arabidopsis thaliana gene FRI (FRIGIDA) exhibits 'adaptive' pleiotropy, producing trait correlations along an axis that results in two adaptive strategies. Derived, low expression FRI alleles confer a 'drought escape' strategy owing to fast growth, low water use efficiency and early flowering. By contrast, a dehydration avoidance strategy is conferred by the ancestral phenotype of late flowering, slow growth and efficient water use during photosynthesis. The dehydration avoidant phenotype was recovered when genotypes with null FRI alleles were transformed with functional alleles. Our findings indicate that the well-documented effects of FRI on phenology result from differences in physiology, not only a simple developmental switch.

Interactive effects of water, light and heat stress on photosynthesis in Fremont cottonwood.

Fremont cottonwood seedlings are vulnerable to water stress from rapid water-table decline during river recession in spring. Water stress is usually cited as the reason for reduced establishment, but interactions of water stress with microclimate extremes are more likely the causes of mortality. We assessed photosynthetic responses of Fremont cottonwood seedlings to water, light and heat stresses, which commonly co-occur in habitats where seedlings establish. Under moderate temperature and light conditions, water stress did not affect photosynthetic function. However, stomatal closure during water stress predisposed Fremont cottonwood leaves to light and heat stress, resulting in greatly reduced photosynthesis beginning at 31 °C versus at 41 °C for well-watered plants. Ontogenetic shifts in leaf orientation from horizontal to vertical, which occur as seedlings mature, reduce heat and light stress, especially during water stress. When compared with naturally occurring microclimate extremes, seedling stress responses suggest that reduced assimilation and photoprotection are common for Fremont cottonwood seedlings on exposed point bars where they establish. These reductions in photosynthesis likely have negative impacts on growth and may predispose young (<90-day-old) seedlings to early mortality during rapid water-table declines. Interactions with heat and light stress are more important in these effects than water stress alone.

Potential and realized nutrient resorption in serpentine and non-serpentine chaparral shrubs and trees.

Low-nutrient adapted species have numerous mechanisms that aid in nutrient conservation. Hypothetically, species adapted to nutrient-poor soils should have tighter internal nutrient recycling, as evidenced by greater resorption. However, literature results are mixed. We suggest methodological factors may limit our understanding of this process. We hypothesized that plants adapted to serpentine soils would be more proficient in resorbing N and P than plants adapted to non-serpentine soils, although there would be differences among functional groups within each soil type. For six growing seasons, we sampled senescent leaf tissue from the dominant and co-dominant shrubs and trees found in serpentine and non-serpentine chaparral communities in the California Coast Range. Our study also explicitly included congener pairs found on both soil types. Most species were highly N proficient, but species adapted to serpentine soils were more P proficient. Surprisingly, two of the three potential N-fixing species were also highly N proficient. Evergreen Quercus congeners were more N proficient than their deciduous congener pairs, although there was no difference in P resorption proficiency. Overall, large inter-annual variation was observed among most species sampled, but at least in some years, maximum potential resorption likely was reached. However, climate (temperature and precipitation) was not strongly correlated with either N or P resorption proficiency. Our data suggest that controlling for phylogeny can aid in interpretation of resorption patterns. More importantly, our study clearly shows that resorption patterns can only be discerned through long-term datasets, of which few exist in the literature.

Characterizing genomic variation of Arabidopsis thaliana: the roles of geography and climate.

Arabidopsis thaliana inhabits diverse climates and exhibits varied phenology across its range. Although A. thaliana is an extremely well-studied model species, the relationship between geography, growing season climate and its genetic variation is poorly characterized. We used redundancy analysis (RDA) to quantify the association of genomic variation [214 051 single nucleotide polymorphisms (SNPs)] with geography and climate among 1003 accessions collected from 447 locations in Eurasia. We identified climate variables most correlated with genomic variation, which may be important selective gradients related to local adaptation across the species range. Climate variation among sites of origin explained slightly more genomic variation than geographical distance. Large-scale spatial gradients and early spring temperatures explained the most genomic variation, while growing season and summer conditions explained the most after controlling for spatial structure. SNP variation in Scandinavia showed the greatest climate structure among regions, possibly because of relatively consistent phenology and life history of populations in this region. Climate variation explained more variation among nonsynonymous SNPs than expected by chance, suggesting that much of the climatic structure of SNP correlations is due to changes in coding sequence that may underlie local adaptation.

Light-stress avoidance mechanisms in a Sphagnum-dominated wet coastal Arctic tundra ecosystem in Alaska.

The Arctic experiences a high-radiation environment in the summer with 24-hour daylight for more than two months. Damage to plants and ecosystem metabolism can be muted by overcast conditions common in much of the Arctic. However, with climate change, extreme dry years and clearer skies could lead to the risk of increased photoxidation and photoinhibition in Arctic primary producers. Mosses, which often exceed the NPP of vascular plants in Arctic areas, are often understudied. As a result, the effect of specific environmental factors, including light, on these growth forms is poorly understood. Here, we investigated net ecosystem exchange (NEE) at the ecosystem scale, net Sphagnum CO2 exchange (NSE), and photoinhibition to better understand the impact of light on carbon exchange from a moss-dominated coastal tundra ecosystem during the summer season 2006. Sphagnum photosynthesis showed photoinhibition early in the season coupled with low ecosystem NEE. However, later in the season, Sphagnum maintained a significant CO2 uptake, probably for the development of subsurface moss layers protected from strong radiation. We suggest that the compact canopy structure of Sphagnum reduces light penetration to the subsurface layers of the moss mat and thereby protects the active photosynthetic tissues from damage. This stress avoidance mechanism allowed Sphagnum to constitute a significant percentage (up to 60%) of the ecosystem net daytime CO2 uptake at the end of the growing season despite the high levels of radiation experienced.

Exploring genetic and expression differences between physiologically extreme ecotypes: comparative genomic hybridization and gene expression studies of Kas-1 and Tsu-1 accessions of Arabidopsis thaliana.

Recent studies have documented remarkable genetic variation among Arabidopsis thaliana accessions collected from diverse habitats. Of particular interest are accessions with putatively locally adapted phenotypes - that is, accessions with attributes that are likely adaptive at their sites of origin. These genotypes may provide insight into the genetic basis of adaptive evolution as well as allow the discovery of genes of ecological importance. We studied the physiology, genome content and gene expression of two physiologically extreme accessions (Tsu-1 from Tsushima, Japan and Kas-1 from Kashmir, India). Our study was conducted under two levels of soil moisture and accompanied by physiological measurements to characterize early responses to soil drying. Genomic hybridizations identified 42,503 single feature polymorphisms (SFP) between accessions, providing an initial screen for genetic differences. Transcript profiling identified a large number (5996) of genes exhibiting constitutive differences in expression including genes involved in many biological pathways. Mild soil drying resulted in only subtle physiological responses but resulted in gene expression changes in hundreds of transcripts, including 352 genes exhibiting differential responses between accessions. Our results highlight the value of genomic studies of natural accessions as well as identify a number of candidate genes underlying physiological differences between Tsu-1 and Kas-1.

Photosynthesis affects following night leaf conductance in Vicia faba.

Night-time stomatal opening in C(3) plants may result in significant water loss when no carbon gain is possible. The objective of this study was to determine if endogenous patterns of night-time stomatal opening, as reflected in leaf conductance, in Vicia faba are affected by photosynthetic conditions the previous day. Reducing photosynthesis with low light or low CO(2) resulted in reduced night-time stomatal opening the following night, irrespective of the effects on daytime stomatal conductance. Likewise, increasing photosynthesis with enriched CO(2) levels resulted in increased night-time stomatal opening the following night. Reduced night-time stomatal opening was not the result of an inability to regulate stomatal aperture as leaves with reduced night-time stomatal opening were capable of greater night-time opening when exposed to low CO(2). After acclimating plants to long or short days, it was found that night-time leaf conductance was greater in plants acclimated to short days, and associated with greater leaf starch and nitrate accumulation, both of which may affect night-time guard cell osmotic potential. Direct measurement of guard cell contents during endogenous night-time stomatal opening will help identify the mechanism of the effect of daytime photosynthesis on subsequent night-time stomatal regulation.

Night-time transpiration can decrease hydraulic redistribution.

C(3) plants dominate many landscapes and are critically important for ecosystem water cycling. At night, plant water losses can include transpiration (E(night)) from the canopy and hydraulic redistribution (HR) from roots. We tested whether E(night) limits the magnitude of HR in a greenhouse study using Artemisia tridentata, Helianthus anomalus and Quercus laevis. Plants were grown with their roots split between two compartments. HR was initiated by briefly withholding all water, followed by watering only one rooting compartment. Under study conditions, all species showed substantial E(night) and HR (highest minus lowest soil water potential [Psi(s)] during a specified diel period). Suppressing E(night) by canopy bagging increased HR during the nightly bagging period (HR(N)) for A. tridentata and H. anomalus by 73 and 33% respectively, but did not affect HR(N) by Q. laevis. Total daily HR (HR(T)) was positively correlated with the Psi(s) gradient between the rooting compartments, which was correlated with light and/or atmospheric vapour pressure deficit (VPDa) the prior day. For A. tridentata, HR(T) was negatively correlated with night-time VPDa. Ecological implications of the impact of E(night) on HR may include decreased plant productivity during dry seasons, altered ecosystem water flux patterns and reduced nutrient cycling in drying soils.

Patterns of nocturnal rehydration in root tissues of Vaccinium corymbosum L. under severe drought conditions.

Although roots in dry soil layers are commonly rehydrated by internal hydraulic redistribution during the nocturnal period, patterns of tissue rehydration are poorly understood. Rates of nocturnal rehydration were examined in roots of different orders in Vaccinium corymbosum L. 'Bluecrop' (Northern highbush blueberry) grown in a split-pot system with one set of roots in relatively moist soil and the other set of roots in dry soil. Vaccinium is noted for a highly branched and extremely fine root system. It is hypothesized that nocturnal root tissue rehydration would be slow, especially in the distal root orders because of their greater hydraulic constraints (smaller vessel diameters and fewer number of vessels). Vaccinium root hydraulic properties delayed internal water movement. Even when water was readily available to roots in the wet soil and transpiration was minimal, it took a whole night-time period of 12 h for the distal finest roots (1st to 4th order) under dry soil conditions to reach the same water potentials as fine roots in moist soil (1st to 4th order). Even though roots under dry soil equilibrated with roots in moist soil, the equilibrium point reached before sunrise was about -1.2 MPa, indicating that tissues were not fully rehydrated. Using a single-branch root model, it was estimated that individual roots exhibiting the lowest water potentials in dry soil were 1st order roots (distal finest roots of the root system). However, considered at the branch level, root orders with the highest hydraulic resistances corresponded to the lowest orders of the permanent root system (3rd-, 4th-, and 5th-order roots), thus indicating possible locations of hydraulic safety control in the root system of this species.

Magnitude of nighttime transpiration does not affect plant growth or nutrition in well-watered Arabidopsis.

Significant water loss occurs throughout the night via partially open stomata in many C(3) and C(4) plant species. Although apparently wasteful in terms of water use, nighttime transpiration (E(night)) is hypothesized to benefit plants by enhancing nutrient supply. We tested the hypothesis that plants with greater E(night) would have improved plant nutrient status and greater fitness, estimated as pre-bolting biomass, for Arabidopsis thaliana. Two very different levels of E(night) were generated in plants by exposing them to high vs low nighttime leaf-to-air vapor pressure deficits (VPD(leaf)) in controlled environment chambers. An assessment of responses of nighttime leaf conductance (g(night)) to VPD(leaf) indicated that E(night) differed by at least 80% between the treatments. This large difference in E(night), imposed over the entire vegetative growth phase of Arabidopsis, had no effect on leaf nutrient content (N, Ca, K) or pre-bolting rosette biomass. The lack of response to differences in E(night) held true for both a high and a low nitrogen (N) treatment, even though the low N treatment decreased leaf N and biomass by 40-60%. The N treatment had no effect on g(night). Thus, higher E(night) did not provide a nutrient or growth benefit to Arabidopsis, even when the plants were N-limited.

Genetic variation in Arabidopsis thaliana for night-time leaf conductance.

Night-time leaf conductance (g(night)) and transpiration may have several adaptive benefits related to plant water, nutrient and carbon relations. Little is known, however, about genetic variation in g(night) and whether this variation correlates with other gas exchange traits related to water use and/or native habitat climate. We investigated g(night) in 12 natural accessions and three near isogenic lines (NILs) of Arabidopsis thaliana. Genetic variation in g(night) was found for the natural accessions, and g(night) was negatively correlated with native habitat atmospheric vapour pressure deficit (VPD(air)), suggesting lower g(night) may be favoured by natural selection in drier habitats. However, there were also significant genetic correlations of g(night) with daytime gas exchange traits expected to affect plant fitness [i.e. daytime leaf conductance, photosynthesis and intrinsic water-use efficiency (WUE(i))], indicating that selection on daytime gas exchange traits may result in indirect selection on g(night). The comparison of three NILs to their parental genotypes identified one quantitative trait locus (QTL) contributing to variation in g(night). Further characterization of genetic variation in g(night) within and among populations and species, and of associations with other traits and native habitats will be needed to understand g(night) as a putatively adaptive trait.

Variable responses of insects to hybrid versus parental sagebrush in common gardens.

Both ecological and genetic mechanisms have been proposed to explain patterns of herbivore attack on interspecific plant hybrids, but distinguishing among them can be difficult in natural hybrid zones. We performed a common-garden experiment to evaluate four genetic hypotheses: dominance, additivity, elevated hybrid susceptibility, and elevated hybrid resistance. Censuses and cage experiments were used to compare insect responses to basin big sagebrush (Artemisia tridentata spp. tridentata), mountain big sagebrush (A. t. vaseyana), and their F2 progeny. After two growing seasons, hybrid shrubs resembled mountain big sagenbrush in size, but were more similar to basin big sagebrush in flower production. Censuses of naturally colonizing insects (the gall midge Rhopalomyia obovata, the bagworm moth Apterona helix, and the aphid Obtusicauda coweni) tended to support the dominance hypothesis: if the insect clearly discriminated between the two parents, its frequency on hybrids closely resembled that on one of the parents. Moreover, colonization of hybrids in all three cases suggested a dominance deviation toward the susceptible parent rather than toward the resistant parent. In contrast to the censuses, cage experiments involving two insects supported the hybrid-susceptibility hypothesis; both survival and growth of the grasshopper Melanoplus sanguinipes and growth of the leaf beetle Trirhabda pilosa were higher on hybrid shrubs than on either parent. Because many secondary compounds have been determined to occur at intermediate concentrations in F2 shrubs, dominance for susceptibility may indicate that insects respond to plant traits (e.g., oviposition stimulants and deterrents) in a threshold manner. Mechanisms underlying increased hybrid susceptibility are less clear, but our experimental design makes environmental explanations (e.g., the plant-stress hypothesis) unlikely. Although we eliminated several confounding factors, our results agree with the conclusion from natural hybrid zones that insect responses to hybrid plants are likely to be idiosyncratic; even congeneric species did not respond similarly to hybrid and parental plants.