A de novo long-read genome assembly of the sacred datura plant (Datura wrightii) reveals a role of tandem gene duplications in the evolution of herbivore-defense response.

The sacred datura plant (Solanales: Solanaceae: Datura wrightii) has been used to study plant-herbivore interactions for decades. The wealth of information that has resulted leads it to have potential as a model system for studying the ecological and evolutionary genomics of these interactions. We present a de novo Datura wrightii genome assembled using PacBio HiFi long-reads. Our assembly is highly complete and contiguous (N50 = 179Mb, BUSCO Complete = 97.6%). We successfully detected a previously documented ancient whole genome duplication using our assembly and have classified the gene duplication history that generated its coding sequence content. We use it as the basis for a genome-guided differential expression analysis to identify the induced responses of this plant to one of its specialized herbivores (Coleoptera: Chrysomelidae: Lema daturaphila). We find over 3000 differentially expressed genes associated with herbivory and that elevated expression levels of over 200 genes last for several days. We also combined our analyses to determine the role that different gene duplication categories have played in the evolution of Datura-herbivore interactions. We find that tandem duplications have expanded multiple functional groups of herbivore responsive genes with defensive functions, including UGT-glycosyltranserases, oxidoreductase enzymes, and peptidase inhibitors. Overall, our results expand our knowledge of herbivore-induced plant transcriptional responses and the evolutionary history of the underlying herbivore-response genes.

Abiotic induction affects the costs and benefits of inducible herbivore defenses in Datura wrightii.

We evaluated the costs and benefits of continuous high-level expression of defenses relative to naturally-induced defenses in field-grown Datura wrightii in the presence and absence of herbivores. We induced D. wrightii plants with monthly applications of the plant hormone methyl jasmonate (MeJA) and assessed levels of inducible proteinase inhibitors (Pins). MeJA application increased Pin production by 124 %, whereas the increase in Pins due to herbivory was more modest (36 %). Pin induction was costly and significantly reduced plant fitness compared to unmanipulated plants both in the presence and absence of herbivores. Although MeJA-treated plants exposed to herbivory suffered significantly less herbivore damage than unmanipulated plants exposed to herbivory, this was not accompanied by a corresponding fitness benefit. In contrast to glasshouse studies in which protected plants never expressed Pins, Pin induction occurred in field-grown plants not treated with MeJA and completely protected from herbivory. Subsequent experiments confirmed that putative herbivore defenses can be induced abiotically in D. wrightii as: 1) Pin levels did not differ significantly between field-grown plants protected from herbivory and plants exposed to chronic herbivory over the full season; and 2) plants exposed to ambient UV-B light in the absence of herbivory expressed low levels of Pins after two wk of exposure, whereas plants protected from UV-B remained uninduced. The costs of induced responses may be relatively easily determined under field conditions, but there may be many inducing agents in the field, and the benefits of induction may be difficult to associate with any single inducing agent.

Ecological role of volatiles produced by plants in response to damage by herbivorous insects.

Plants often release a blend of volatile organic compounds in response to damage by herbivorous insects that may serve as cues to locate those herbivores by natural enemies. The blend of compounds emitted by plants may be more variable than is generally assumed. The quantity and the composition of the blends may vary with the species of the herbivore, the plant species and genotype within species, the environmental conditions under which plants are grown, and the number of herbivore species attacking the plant. Although it is often assumed that induced emission of these compounds is an adaptive tactic on the part of plants, the evidence that such responses minimize fitness losses of plants remains sparse because the necessary data on plant fitness rarely have been collected. The application of techniques of evolutionary quantitative genetics may facilitate the testing of widely held hypotheses about the evolution of induced production of volatile compounds under natural conditions.

The impact of rapid evolution on population dynamics in the wild: experimental test of eco-evolutionary dynamics.

Rapid evolution challenges the assumption that evolution is too slow to impact short-term ecological dynamics. This insight motivates the study of 'Eco-Evolutionary Dynamics' or how evolution and ecological processes reciprocally interact on short time scales. We tested how rapid evolution impacts concurrent population dynamics using an aphid (Myzus persicae) and an undomesticated host (Hirschfeldia incana) in replicated wild populations. We manipulated evolvability by creating non-evolving (single clone) and potentially evolving (two-clone) aphid populations that contained genetic variation in intrinsic growth rate. We observed significant evolution in two-clone populations whether or not they were exposed to predators and competitors. Evolving populations grew up to 42% faster and attained up to 67% higher density, compared with non-evolving control populations but only in treatments exposed to competitors and predators. Increased density also correlates with relative fitness of competing clones suggesting a full eco-evolutionary dynamic cycle defined as reciprocal interactions between evolution and density.

Production of induced volatiles by Datura wrightii in response to damage by insects: effect of herbivore species and time.

Natural enemies of herbivorous insects utilize numerous chemical cues to locate and identify their prey. Among these, volatile plant compounds produced after attack by herbivores may play a significant role (hereafter herbivore-induced plant volatiles or HIPVs). One unresolved question is whether the composition of the volatile cue blends induced by different herbivore species differ consistently enough to indicate not only that the plants are damaged by herbivores but also the identity of the herbivore species causing the damage. We studied HIPV production in the undomesticated plant species Datura wrightii in the laboratory when damaged by either of two leaf-chewing herbivore species, Lema daturaphila or Manduca sexta, or when damaged by L. daturaphila and the piercing-sucking bug, Tupiocoris notatus, or both L. daturaphila and T. notatus, for 24 hr. HIPV production was monitored 1 d before induction, the day of induction, and for 7 d after induction. In all experiments, both the quantities and composition of the HIPV blends varied with the time since induction as different components reached peak production at different times after induction. HIPV blends did not differ consistently with the herbivore species causing the damage. For plants damaged by both L. daturaphila and T. notatus, greater amounts of HIPVs were produced than by plants damaged by either species alone, but the amounts did not differ from that predicted as the sum from damage inflicted by each herbivore species independently. The HIPVs of D. wrightii are a general rather than specific indicator of damage by herbivores. Because generalist predators are the most abundant natural enemies in this system, general cues of herbivore damage may be all that are required to facilitate the discovery by predators of plants damaged by any of several suitable prey species.

Production of herbivore-induced plant volatiles is constrained seasonally in the field but predation on herbivores is not.

Natural enemies of herbivorous insects utilize numerous cues to locate and identify their prey. One class of such cues is volatile organic compounds (VOCs) often produced by plants after attack by herbivores (hereafter herbivore-induced plant volatiles or HIPVs). Under simplified laboratory conditions, natural enemies often make clear choices between different HIPV blends, but such clear choices may be more difficult in more complex field environments. We studied the impact of VOC production by the undomesticated species, Datura wrightii on predation of eggs and larvae of Lema daturaphila by the omnivore, Geocoris pallens in the field. HIPV production in D. wrightii is developmentally and seasonally constrained to the early stages of plant growth even though L. daturaphila and G. pallens inhabit plants throughout the plant's growing season. We, therefore, asked if predation of L. daturaphila by G. pallens might be similarly constrained seasonally. Higher levels of VOC production were associated with higher levels of predation throughout the growing season, and the greater quantities of VOC production in May caused greater increases in predation than did VOC production later in the season (June-September). However, predation in the absence of VOC production ranged from 60-70% in June-September compared to only 14% in May, probably because plants were already colonized by predators later in the season. High levels of VOCs in response to herbivore damage by D. wrightii therefore may aid in the discovery of herbivore-damaged plants early in the season but the seasonal decline in VOC production does not limit predation of L. daturaphila by G. pallens later in the season.

Ontogeny and season constrain the production of herbivore-inducible plant volatiles in the field.

Herbivores may induce plants to produce an array of volatile organic compounds (herbivore-induced plant volatiles, or HIPVs) after damage, and some natural enemies of herbivores are attracted by those HIPVs. The production of HIPVs by the undomesticated species Datura wrightii was quantified in response to damage by its natural community of herbivores or the plant hormone methyl jasmonate (MeJA) over plant's 6-month growing season. Patterns of HIPV production were compared to the seasonal abundance of D. wrightii's two most abundant herbivores, the chrysomelid beetle Lema daturaphila and the mirid bug Tupiocoris notatus, and their shared generalist predator, the lygaeid bug Geocoris pallens. HIPV production was especially high in the spring, when plants were growing vegetatively, but HIPV production declined after plants began to flower and produce fruit, and these volatiles no longer were inducible by September. The composition of the HIPV blends also changed seasonally. HIPV production and composition were partially restored by "rejuvenating" plants back to the vegetative growth stage independently of season by cutting them back and allowing them to resprout and regrow vegetatively. HIPV production of D. wrightii in the field is limited to the earlier ontogenetic stages of growth, despite the fact that both herbivores and their shared natural enemy inhabited plants throughout the full season. The adaptive value of HIPV production in D. wrightii may be constrained by plant ontogeny to the vegetative stages of plant growth.

Herbivore-mediated negative frequency-dependent selection underlies a trichome dimorphism in nature.

Negative frequency-dependent selection (NFDS) has been shown to maintain polymorphism in a diverse array of traits. The action of NFDS has been confirmed through modeling, experimental approaches, and genetic analyses. In this study, we investigated NFDS in the wild using morph-frequency changes spanning a 20-year period from over 30 dimorphic populations of Datura wrightii. In these populations, plants either possess glandular (sticky) or non-glandular (velvety) trichomes, and the ratio of these morphs varies substantially among populations. Our method provided evidence that NFDS, rather than drift or migration, is the primary force maintaining this dimorphism. Most populations that were initially dimorphic remained dimorphic, and the overall mean and variance in morph frequency did not change over time. Furthermore, morph-frequency differences were not related to geographic distances. Together, these results indicate that neither directional selection, drift, or migration played a substantial role in determining morph frequencies. However, as predicted by negative frequency-dependent selection, we found that the rare morph tended to increase in frequency, leading to a negative relationship between the change in the frequency of the sticky morph and its initial frequency. In addition, we found that morph-frequency change over time was significantly correlated with the damage inflicted by two herbivores: Lema daturaphila and Tupiochoris notatus. The latter is a specialist on the sticky morph and damage by this herbivore was greatest when the sticky morph was common. The reverse was true for L. daturaphila, such that damage increased with the frequency of the velvety morph. These findings suggest that these herbivores contribute to balancing selection on the observed trichome dimorphism.

Costs of glandular trichomes in Datura wrightii: a three-year study.

Models accounting for genetic variation for resistance to herbivores within plant populations often postulate a balance between the costs of that resistance and its benefits. The production of glandular trichomes by Datura wrightii was shown to be costly in a previous one-year study because plants producing glandular trichomes (sticky plants), a factor conferring resistance to some insect herbivores, also produced 45% fewer seeds than plants producing nonglandular trichomes (velvety plants) when grown in a common garden. Because sticky plants tended to be larger than velvety plants but produced fewer seed capsules, we postulated an allocation trade-off in which velvety plants are more reproduction-dominated whereas sticky plants are more growth-dominated. If a greater commitment to vegetative growth eventually allows sticky plants to compensate for reduced seed production, we would expect a reduction or elimination of the cost of resistance over time in this perennial plant. We monitored growth, survival, and seed production of plants from defined crosses of local populations for three years in a common garden when exposed to and protected from herbivores, and with and without supplemental water. The majority of plants exposed to herbivores had died by the end of the study. We used standard life-table methods to determine the net reproductive rate (R0) and the finite rate of increase (lambda) of plants of each trichome type. After three years, when plants were protected from herbivores, sticky plants were 187-245% larger than velvety plants, depending upon irrigation treatment, but sticky plants continued to be less efficient in producing seeds per unit of canopy volume. Even though the total seed production of sticky plants eventually equaled that of velvety plants, the advantage of earlier reproduction by velvety plants increased lambda by 55-230% over that of sticky plants, depending upon herbivore and irrigation treatment. Exposure to herbivores reduced lambda by 69-83%, depending upon plant type and irrigation treatment, whereas supplemental irrigation increased lambda by 29-175%, depending upon plant type and exposure to herbivores. Although there was a large allocation trade-off between growth and reproduction, the benefits of such a trade-off did not emerge before most plants were killed by herbivores. The cost of producing glandular trichomes strictly for herbivore resistance continued to exceed its benefits, and in the absence of other, unmeasured benefits from the suite of life-history characters associated with glandular trichome production, natural selection is expected to eliminate this costly resistance trait from D. wrightii populations.

Structure of a geranyl-alpha-pyrone from Mimulus aurantiacus leaf resin.

A previously reported but misidentified geranyl-alpha-pyrone, in addition to six known compounds, was isolated from the leaf resin of Mimulus aurantiacus. HMBC NMR analyses of the geranyl-alpha-pyrone resolved uncertainties in the site of attachment of the two side chains and necessitated a revision of the previously reported structure. This compound is shown to be 3-geranyl-4-hydroxy-6-(2-hydroxypropyl)-2-pyrone.

Variation in fruit size and susceptibility to seed predation among and within populations of the cocklebur, Xanthium strumarium L.

Burr size is the major factor affecting variation in the intensity of predation by two species of insect on the seeds of the cocklebur, Xanthium strumarium. Mean burr size varied among 10 adjacent local populations studied over three years, as did intensity of seed predation. Seed predation was more intense in populations with low mean burr length and declined linearly with increasing burr length under field and experimental conditions. Seed predation thus is a selective factor influencing the evolution of both burr size and correlated protective characteristics such as burr spine length and wall thickness. As in some other plants, morphological rather than chemical features appear to pose the major barrier to attack by host-specific seed predators. The advantage of more highly developed tissues protecting seeds may occur at the expense of total seed production.

Different effects of variation in Xanthium strumarium L. (Compositae) on two insect seed predators.

To determine the relative importance of variation in several plant characters on susceptibility to herbivores, we examined patterns of seed predation by two monophagous insect species and patterns of variation in ten populations of the cocklebur, Xanthium strumarium. Multiple regression analysis disclosed that one seed predator was most influenced by plant chemical variation, the other was significantly influenced by both chemical and morphological variation, but variation in yet another character, general burr size, was most important in conferring resistance to both insects simultaneously. The plant populations differed most in this character. Although many of the plant characters were correlated with each other, those important in determining susceptibility to each insect species were uncorrelated and independent of those conferring resistance to both insects simultaneously.These results imply that ecological similar herbivores may be influenced by different aspects of plant variation, and that predictions of evolutionary responses of local plant populations to herbivory may require knowledge of the structure of local herbivore communities and the dynamics of their establishment.

Foliar terpenoids in Tsuga species and the fecundity of scale insects.

The relationship between the reproductive success of two Japanese scale insects, Fiorinia externa Ferris and Nuculaspis tsugae (Marlatt) (Homoptera: Diaspididae) and the concentrations of 15 terpenoids in needles of Tsuga sieboldii, the Japanese host, and T. canadensis, the North American host, was investigated during 1981 and 1982 in a field plot of 8-year-old trees in New Haven, CT, USA. Both scales produced significantly more eggs per female on T. sieboldii than on T. canadensis. Stepwise multiple regression analyses indicated that the variation in fecundity within both scales was strongly associated with variation in the terpenoid profile between tree species.General patterns of phytochemical variation between the two Tsuga species based on differences in the concentration of terpenoids having similar chemical structures were revealed by the multivariate statistical technique, principal components analysis. The volatile leaf oil profile of T. sieboldii was relatively richer in terpene alcohols, while that of T. canadensis was relatively richer in terpene hydrocarbons and terpene acetates. The individual terpenoids were then assigned to one of five groups based on chemical structure and regression analyses were repeated; fecundity of both scales increased with increasing concentration of terpenoid alcohols. Fecundity of F. externa was negatively associated with the relative concentration of acyclic terpenes but the opposite was true for N. tsugae. Analysis of foliar terpenoids may provide a basis for predicting the relative susceptibility of Tsuga species to attack by F. externa and N. tsugae.

Variation in herbivore and methyl jasmonate-induced volatiles among genetic lines of Datura wrightii.

Many plant species produce volatile organic compounds after being damaged by herbivores. The production of volatiles also may be induced by exposing plants to the plant hormone, jasmonic acid, or its volatile ester, methyl jasmonate. This study addresses the induction of the production volatile organic compounds among genetic lines of Datura wrightii. Within populations, some plants produce glandular trichomes, whereas others produce nonglandular trichomes, and trichome phenotype is controlled by a single dominant gene. Glandular trichomes not only confer resistance to some herbivorous insects, but they also inhibit many natural enemies of those herbivores. Because of the potential benefit of natural enemies that use volatile cues to find individuals of the non-glandular phenotype, it is reasonable to ask if plants of D. wrightii that differ in trichome morphology might produce different blends of volatile compounds. Volatile compounds were collected from eight genetic lines of plants that had been backcrossed for three generations. Volatiles were collected from pairs of sibling plants before and after insect damage or treatment with methyl jasmonate. Within each pair, one sib expressed glandular trichomes and the other expressed nonglandular trichomes. Overall, plants produced an array of at least 17 compounds, most of which were sesquiterpenes. Total production of volatiles increased from 3.9- to 16.2-fold among genetic lines after insect damage and from 3.6- to 32-fold in plants treated with methyl jasmonate. The most abundant compound was (E)-beta-caryophyllene. This single compound comprised from 17 to 59% of the volatiles from insect-damaged plants and from 24 to 88% of the volatiles from plants treated with methyl jasmonate, depending upon genetic line. The production of (E)-beta-caryophyllene by the original male parents of the eight genetic lines was significantly related to the mean production of their third-generation backcross progeny indicating that the variation in the production of (E)-beta-caryophyllene was inherited. Blends did not differ qualitatively or quantitatively between sibs expressing glandular or nonglandular trichomes.

Constitutive and jasmonate-inducible traits of Datura wrightii.

Plants in the family Solanaceae possess numerous traits that are induced from damage from herbivores. Many of these also can be induced by exposing plants to the plant hormone jasmonic acid or its volatile ester methyl jasmonate. Datura wrightii (Solanaceae) is dimorphic for leaf trichome morphology in most southern California populations. Trichome phenotype is governed by a single gene, and the glandular trichome condition is dominant and under developmental control. This study addressed two major objectives. The first was to determine if mature plants with glandular or nonglandular trichomes responded differentially to methyl jasmonate. The second objective was to determine if exposure of seedlings to methyl jasmonate during the period of trichome differentiation altered either the phenotype or the density of trichomes that mature plants expressed. Methyl jasmonate induced from 200 to 800 microg/ml of proteinase inhibitor activity and increased the activity of polyphenol oxidase by more than threefold depending on the experiment. These increases did not differ significantly between plants expressing glandular or nonglandular trichomes. Methyl jasmonate exposure did not increase the activity of peroxidase or the concentration of scopolamine or hyoscyamine, the two major alkaloids of Datura. Exposure to methyl jasmonate during trichome differentiation did not affect either the final trichome phenotype or the density of either type of trichome, but did increase the production of acylsugars in glandular trichomes by 44%. Because trichome phenotype was not inducible, and because both trichome phenotypes showed similar increases in proteinase inhibitors and polyphenol oxidase activity, the methyl-jasmonate-inducible responses of D. wrightii are independent of trichome phenotype in D. wrightii.

Biological activity of acyl glucose esters from Datura wrightii glandular trichomes against three native insect herbivores.

Datura wrightii is dimorphic for leaf trichome type in southern California. "Sticky" plants produce glandular trichomes that secrete acylsugars, whereas velvety plants produce nonglandular trichomes. Glandular trichomes confer resistance to some potential insect herbivores and are associated with reduced feeding in the field by two native coleopteran herbivores: the tobacco flea beetle, Epitrix hirtipennis, and a weevil, Trichobaris compacta. In contrast, another native beetle, Lema daturaphila, damages sticky and velvety plants similarly in the field. A series of choice and no-choice "ester removal" and "ester addition" feeding experiments were performed in the laboratory to evaluate the role of acylsugars in feeding by all three insect species. Consumption of sticky leaves after their esters were removed by washing was compared to consumption of unwashed sticky leaves and velvety leaves in ester removal experiments. Consumption of velvety leaves was measured after acylsugars were applied to those leaves in controlled amounts in the ester addition experiments. Consumption by E. hirtipennis was reduced by acylsugars in all experiments. Consumption by T. compacta was reduced by acylsugars in the ester removal experiments, but not in the ester addition experiments. The location of the acylsugars at the tip of a long trichome, rather than simply on the leaf surface, may be an important component of the biological activity of acylsugars against T. compacta in nature. Consumption by L. daturaphila was not significantly reduced by acylsugars in any experiment. The acylsugars caused no significant mortality of any of the three insect species.

Indirect cost of a defensive trait: variation in trichome type affects the natural enemies of herbivorous insects on Datura wrightii.

The costs and benefits of defensive traits in plants can have an ecological component that arises from the effect of defenses on the natural enemies of herbivores. We tested if glandular trichomes in Datura wrightii, a trait that confers resistance to several species of herbivorous insects, impose an ecological cost by decreasing rates of predation by the natural enemies of herbivores. For two common herbivores of D. wrightii, Lema daturaphila and Tupiocoris notatus, several generalized species of natural enemies exhibited lower rates of predation on glandular compared to non-glandular plants. Lower rates of predation were associated with reductions in the residence time and foraging efficiency of natural enemies on plants with glandular trichomes, but not with direct toxic effects of glandular exudate. Our results suggest that the benefit of resistance to herbivores conferred by glandular trichomes might be offset by the detrimental effect of this trait on the natural enemies of herbivores, and that the fitness consequences of this trichome defense might depend on the composition and abundance of the natural-enemy community.

GENETIC VARIATION IN PLANT-INSECT ASSOCIATIONS: SURVIVAL OF LEPTINOTARSA DECEMLINEATA POPULATIONS ON SOLANUM CAROLINENSE.

Populations of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), from the east coast of the United States differ in their ability to survive on a wild host, Solanum carolinense (Solanaceae), but not on their most important cultivated host, Solanum tuberosum. On the wild host, the North Carolina population survived best, while populations from Virginia, New Jersey, and Connecticut exhibited uniformly low survival. Formal genetic studies of populations from Connecticut and North Carolina demonstrated heritable variation in the ability to survive on S. carolinense both between and within populations; the North Carolina population had the higher heritability for this trait. Overall, there was no genetic variation between populations or within the North Carolina population for survival on S. tuberosum, but such variation existed within the Connecticut population. Hybrids and backcrosses between these two lines all survived at intermediate levels, although survivorship did not appear to be inherited additively. Differences in survival were greater than differences in adult weight at emergence and development time of the survivors. Leptinotarsa decemlineata was first reported from North Carolina less than 100 years ago. The rapid expansion of L. decemlineata's host range in North Carolina is attributed to the poor synchrony between the insect and S. tuberosum compared to more northerly locations. In contrast to the prediction of a strong negative correlation in fitness on different host species, the ability of L. decemlineata to survive on S. carolinense was not correlated with that on S. tuberosum. Adult weight and female development time were significantly positively correlated across hosts. Our results are in accord with most previous studies in which strong negative correlations in fitness of specialized phytophagous insects feeding on different hosts were expected, sought, but not found.

Spectral properties, gas exchange, and water potential of leaves of glandular and non-glandular trichome types in Datura wrightii (Solanaceae).

Plant trichomes commonly serve a role in mechanical and chemical defence against herbivores, but may also have the potential to alter physiology by reducing the amount of light absorbed by leaves, lowering temperatures, and reducing water loss. Populations of Datura wrightii Regel in southern California produce 'sticky' plants with glandular trichomes and 'velvety' plants bearing non-glandular trichomes. Because stickiness is inherited as a dominant Mendelian trait, and the proportions of sticky plants vary among populations with the moisture availability of their environment, there may be some ecophysiological differences between trichome types that contribute to their ability to survive in a particular geographic location. To examine the possible physiological significance of trichome variation, we measured the spectral properties, midday gas-exchange rates, and water potentials of D. wrightii leaves from sticky and velvety plants growing in a field experiment. The differences in leaf reflectance (0.9%) and absorptance (1.3%) of photosynthetically active radiation (PAR) between trichome types are too small to have any direct physiologically significant effect. Simulations of leaf temperatures based on the difference in leaf absorptances reveal that leaf temperature would be no more than 1°C lower in velvety compared to sticky plants. Gas-exchange measurements revealed no significant difference between types in their transpiration rates or stomatal conductances. In this case, trichome variation may be more important to plant defenses than to physiology.