Genetically based latitudinal variation in Artemisia californica secondary chemistry

Steep climatic gradients may select for clinal adaptation in plant functional traits with implications for interspecific interactions and response to future climate change. Terpenes are common in Mediterranean environments and mediate plant interactions with both the abiotic and biotic environment, including herbivores. Clines in traits such as terpenes have received much attention because they are linked to plant fitness and experience strong selection from the abiotic and biotic environment. In this study, we tested for intraspecific variation in Artemisia californica terpene chemistry in a common garden of plants sourced from populations spanning a large precipitation gradient (6° latitude) and grown in treatments of high and low precipitation. We found genetic variation in terpene richness, diversity, concentration and composition among A. californica populations spanning this species’ range. Of these traits, terpene composition and monoterpene concentration varied clinally with respect to source site latitude. Regarding terpene composition, pairwise dissimilarity among populations increased in parallel with geographic distance between source sites. At the same time, monoterpene concentration decreased monotonically from plants of southern origin (source sites with high temperature, aridity, and precipitation variability) to plants of northern origin. Our precipitation manipulation suggests that phenotypic selection by precipitation may underlie this clinal variation in monoterpene concentration, and that monoterpene concentration and other aspects of terpene chemistry are not phenotypically plastic. In summary, this study provides novel evidence for a genetically based latitudinal cline in plant secondary chemistry and suggests that adaptation to a key aspect of the abiotic environment may contribute to this intraspecific variation. Accordingly, changes in terpene chemistry under projected future climates will likely occur solely through the relatively slow process of adaptation, with important consequences for plant interactions with the abiotic environment and a diverse community of associates.     

Plant defence as a complex and changing phenotype throughout ontogeny

Background and Aims Ontogenetic changes in anti-herbivore defences are common and result from variation in resource availability and herbivore damage throughout plant development. However, little is known about the simultaneous changes of multiple defences across the entire development of plants, and how such changes affect plant damage in the field. The aim of this study was to assess if changes in the major types of plant resistance and tolerance can explain natural herbivore damage throughout plant ontogeny.Methods An assessment was made of how six defensive traits, including physical, chemical and biotic resistance, simultaneously change across the major transitions of plant development, from seedlings to reproductive stages of Turnera velutina growing in the greenhouse. In addition, an experiment was performed to assess how plant tolerance to artificial damage to leaves changed throughout ontogeny. Finally, leaf damage by herbivores was evaluated in a natural population.Key Results The observed ontogenetic trajectories of all defences were significantly different, sometimes showing opposite directions of change. Whereas trichome density, leaf toughness, extrafloral nectary abundance and nectar production increased, hydrogen cyanide and compensatory responses decreased throughout plant development, from seedlings to reproductive plants. Only water content was higher at the intermediate juvenile ontogenetic stages. Surveys in a natural population over 3 years showed that herbivores consumed more tissue from juvenile plants than from younger seedlings or older reproductive plants. This is consistent with the fact that juvenile plants were the least defended stage.Conclusions The results suggest that defensive trajectories are a mixed result of predictions by the Optimal Defence Theory and the Growth–Differentiation Balance Hypothesis. The study emphasizes the importance of incorporating multiple defences and plant ontogeny into further studies for a more comprehensive understanding of plant defence evolution.     

Ontogenetic and temporal trajectories of chemical defence in a cyanogenic eucalypt

Many studies have shown that similarly aged plants within a species or population can vary markedly in the concentration of defence compounds they deploy to protect themselves from herbivores. Some studies have also shown that the concentration of these compounds can change with development, but no empirical research has mapped such an ontogenetic trajectory in detail. To do this, we grew cyanogenic Eucalyptus yarraensis seedlings from three half-sibling families under constant glasshouse conditions, and followed their foliar cyanogenic glycoside (prunasin) concentration over time for 338 days after sowing (DAS). Plants in all families followed a similar temporal pattern. Plants increased in foliar prunasin concentration from a very low level (10 μg cyanide (CN) equivalents g⁻¹) in their first leaves, to a maximum of, on average, 1.2 mg CN g⁻¹ at about 240 DAS. From 240 to 338 DAS, prunasin concentration gradually decreased to around 0.7 mg CN g⁻¹. Significant differences between families in maximum prunasin concentration were detected, but none were detected in the time at which this maximum occurred. In parallel with these changes in prunasin concentration, we detected an approximately linear increase in leaf mass per unit leaf area (LMA) with time, which reflected a change from juvenile to adult-like leaf anatomy. When ontogenetic trajectories of prunasin against LMA were constructed, we failed to detect a significant difference between families in the LMA at which maximum prunasin concentration occurred. This remarkable similarity in the temporal and ontogenetic trajectories between individuals, even from geographically remote families, is discussed in relation to a theoretical model for ontogenetic changes in plant defence. Our results show that ontogeny can constrain the expression of plant chemical defense and that chemical defense changes in a nonlinear fashion with ontogeny.     

Early ontogenetic trajectories vary among defence chemicals in seedlings of a fast‐growing eucalypt

Ontogenetic changes in leaf chemistry can affect plant–herbivore interactions profoundly. Various theoretical models predict different ontogenetic trajectories of defence chemicals. Empirical tests do not consistently support one model. In Eucalyptus nitens, a fast‐growing tree, we assessed early developmental changes to seedlings, in foliage concentrations of nitrogen and the full suite of known secondary (defence) chemicals. This included the terpene, α‐pinene, whose impact on marsupial herbivory is unknown. To test for the influence of abiotic conditions on the ontogenetic trajectories we overlaid a nutrient treatment. Ontogenetic trajectories varied among compounds. Sideroxylonals and cineole were barely detected in very young seedlings, but increased substantially over the first 200 days. Total phenolic concentration increased fourfold over this time. In contrast, α‐pinene concentration peaked within the first 60 days and again between 150 and 200 days. Nutrients altered the degree but not the direction of change of most chemicals. A shorter trial run at a different season showed qualitatively similar patterns, although α‐pinene concentration started very high. We investigated the effect of detected levels of α‐pinene and cineole on food intake by two mammalian herbivores, common brushtail possums (Trichosurus vulpecula) and red‐bellied pademelons (Thylogale billardierii). Under no‐choice conditions neither terpene reduced intake; but with a choice, possums preferred α‐pinene to cineole. The ontogenetic trajectories of most compounds were therefore consistent with models that predict an increase as plants develop. Published data from later developmental stages in E. nitens also confirm this pattern. α‐Pinene, however, was the only secondary compound found at significant levels in very young seedlings; but it did not constrain feeding by marsupial herbivores. Models must allow for different roles of defensive secondary chemicals, presumably associated with different selective pressures as plants age, which result in different ontogenetic trajectories.     

Quantitative trait loci for foliar terpenes in a global eucalypt species

Terpenes are a diverse group of plant secondary metabolites that mediate a plethora of ecological interactions in many plant species. Despite increasing research into the genetic control of important adaptive traits in some plant species, the genetic control of terpenes in forest tree species is still relatively poorly studied. In this study, we use quantitative genetic and quantitative trait loci (QTL) analysis to investigate the genetic control of foliar terpenes in an ecologically and commercially important eucalypt species, Eucalyptus globulus. We show a moderate to high within-family broad-sense heritability and significant genetic basis to the variation in 14 of the 16 terpenes assayed. This is the first report of QTL for terpenes in this species. Eleven QTL influenced the terpenes overall. One QTL on linkage group 6 affected six of the seven different sesquiterpenes assayed (plus one monoterpene), which, in combination with highly significant correlations between these compounds, argues that their variation is influenced by a QTL with pleiotropic effect early in the biosynthetic pathway. We examine the homology of these QTL to those found in a closely related eucalypt, Eucalyptus nitens, and provide evidence that both common and unique QTL influence terpene levels.     

Facing herbivory as you grow up: the ontogeny of resistance in plants

As plants develop from seeds to seedlings, juveniles and mature stages, their ontogeny can constrain the expression of resistance to herbivore damage. Nevertheless, ecological and evolutionary theories regarding interactions between plants, herbivores and their natural enemies are largely based on observations and experiments conducted at a single ontogenetic stage. Owing to resource allocation and architectural constraints in plants, and the influence of herbivore foraging behavior, resistance to herbivores is likely to change during plant development. We propose that such changes are likely to occur in a non-linear fashion and suggest that the role of ontogeny should be incorporated as an important factor in new syntheses of plant defense theory.     

Linking deer browsing and terpene production among genetic identities in Chamaecyparis nootkatensis and Thuja plicata (Cupressaceae)

To investigate whether differential herbivore browsing reflects genetic variation in plant defense expression, variation in needle terpenes and damage caused by black-tailed deer (Odocoileus hemionus) was analyzed on yellow-cedar (Chamaecyparis nootkatensis) and western redcedar (Thuja plicata). In a 100-genet yellow-cedar population, three genets that were heavily browsed and had extremely low levels of monoterpenes (0-0.36% dry matter), sesquiterpenes, and diterpenes were compared to unbrowsed genets (0.85-3.83% monoterpenes in dry matter). These differences were maintained in individuals protected from browsing, suggesting genetically based variation in constitutive terpene production. In western redcedar, heavily browsed trees had significantly lower total monoterpene concentrations (1.69% dry matter) than lightly browsed trees (3.32% dry matter). One heavily browsed tree expressed no monoterpenes. No differences were found for diterpenes. In both species, the genotypes with extremely low monoterpene concentrations came from the same open-pollinated families.     

Ontogenetic patterns in the mechanisms of tolerance to herbivory in Plantago

Background and Aims

Herbivory and plant defence differ markedly among seedlings and juvenile and mature plants in most species. While ontogenetic patterns of chemical resistance have been the focus of much research, comparatively little is known about how tolerance to damage changes across ontogeny. Due to dramatic shifts in plant size, resource acquisition, stored reserves and growth, it was predicted that tolerance and related underlying mechanisms would differ among ontogenetic stages.

Methods

Ontogenetic patterns in the mechanisms of tolerance were investigated in Plantago lanceolata and P. major (Plantaginaceae) using the genetic sib-ship approach. Pot-grown plants were subjected to 50 % defoliation at the seedling, juvenile and mature stages and either harvested in the short-term to look at plasticity in growth and photosynthesis in response to damage or allowed to grow through seed maturation to measure phenology, shoot compensation and reproductive fitness.

Key Results

Tolerance to defoliation was high in P. lanceolata, but low in P. major, and did not vary among ontogenetic stages in either species. Mechanisms underlying tolerance did vary across ontogeny. In P. lanceolata, tolerance was significantly related to flowering (juveniles) and pre-damage shoot biomass (mature plants). In P. major, tolerance was significantly related to pre-damage root biomass (seedlings) and induction of non-photochemical quenching, a photosynthetic parameter (juveniles).

Conclusions

Biomass partitioning was very plastic in response to damage and showed associations with tolerance in both species, indicating a strong role in plant defence. In contrast, photosynthesis and phenology showed weaker responses to damage and were related to tolerance only in certain ontogenetic stages. This study highlights the pivotal role of ontogeny in plant defence and herbivory. Additional studies in more species are needed to determine how seedlings tolerate herbivory in general and whether mechanisms vary across ontogeny in consistent patterns.     

Every plant for himself; the effect of a phenolic monoterpene on germination and biomass of Thymus pulegioides and T. serpyllum

Thyme plants are known for their production of aromatic oils, whose main component is terpenes. The plants leach terpenes to their surroundings and thereby affect the seed germination and biomass of associated plants, but also potentially themselves. A variation in the dominant terpenes produced by thyme plants is found both within and among species. In Denmark two thyme species (Thymus pulegioides and T. serpyllum) are naturally occurring. The essential oil of T. pulegioides in Denmark is mainly dominated by one monoterpene; ‘carvacrol’. In contrast, the essential oil of T. serpyllum constitutes a mix of two–four different types of terpenes, both mono‐ and sesqui‐terpenes. As the effects of terpenes on plant performance can vary with the type of terpene, and in order to study species‐specific responses, we examined how the dominating T. pulegioides monoterpene ‘carvacrol’ affected germination and growth of both T. pulegioides and T. serpyllum. We compared the performance of seeds and seedlings of both thyme species on soil treated with carvacrol versus control soil. We found no effect of treatment on germination, but we detected a highly significant effect of treatment on seedling biomass. For both thyme species, seedling biomass was significantly higher on terpene soil compared to control soil, suggesting a general adaptation to the presence of terpenes in the soil for both thyme species. Moreover, while no difference in seedling biomass between species on control soil was found, T. pulegiodes seedlings were significantly larger than T. serpyllum when grown on soil treated with its ‘home’ terpene, suggesting an additional species specific response. Dividing the biomass into aboveground and root biomass showed that the increased biomass on terpene‐soil was due to increased aboveground biomass, whereas no difference in root biomass was detected among treatments and species. We discuss whether this response may be caused by an adaptation to a predictable terpene‐mediated alteration in nitrogen‐availability.     

Stage-structured ontogeny in resource populations generates non-additive stabilizing and de-stabilizing forces in populations and communities

Demographic heterogeneity influences how populations respond to density dependent intraspecific competition and trophic interactions. Distinct stages across an organism's development, or ontogeny, are an important example of demographic heterogeneity. In consumer populations, ontogenetic stage structure has been shown to produce categorical differences in population dynamics, community dynamics and even species coexistence compared to models lacking explicit ontogeny. The study of consumer–resource interactions must also consider the ontogenetic stage structure of the resource itself, particularly plants, given their fundamental role at the basis of terrestrial food webs. We incorporate distinct ontogenetic stages of plants into an adaptable multi-stage consumer–resource modeling framework that facilitates studying how stage specific consumers shape trophic dynamics at low trophic levels. We describe the role of density dependent demographic rates in mediating the dynamics of stage-structured plant populations. We then investigate how these demographic rates interact with consumer pressure to influence stability and coexistence in multiple stage-specific consumer–resource interactions. Results detail how density dependent effects across distinct ontogenetic stages in plant development produce non-additivity in the drivers of dynamic stability both in single populations and in consumer–resource settings, challenging the ubiquity of certain traditional ecological dynamic paradigms. We also find categorical differences in the population variability induced by herbivores consuming separate plant stages. Consumer–resource models, such as plant–herbivore interactions, often average out demographic heterogeneity in populations. Here, we show that explicitly including plant demographic heterogeneity through ontogeny yields distinct dynamic expectations for both plants and herbivores compared to traditional consumer–resource formulations. Our results indicate that efforts to understand the demographic effect of herbivores on plant populations may need to also consider the effects of plant demographics on herbivores and the reciprocal relationship between them.     

Evolution of adaptation through allometric shifts in a marine snail

Variation in ontogenetic development among individuals may be a major contributor to morphological variation within species. Evolution of different growth trajectories might, for example, evolve as a response to varying ecological contexts of individuals living in different environments, or by life-stage or gender differences. The intertidal periwinkle Littorina saxatilis is strongly polymorphic in shell shape. We compared ontogenetic trajectories between life stages, local populations, and sexes to understand how different morphological end points are reached during ontogeny and what might cause these differences. Applying landmark-based geometric morphometrics, we captured shell shape variation for four Swedish populations of this species. We also derived a method to visualize ontogenetic trajectories described by the relationship of size to the multivariate shape space. We found that growth trajectories differed between individuals living in different habitats, as well as between sexes and maturity stages. Males living on rocky cliffs grew isometrically throughout life, whereas females from the same habitat switched from isometric growth as juveniles to allometric growth as adults. In contrast, males and females living on boulders grew allometrically as juveniles but changed to isometric growth at sexual maturity. Thus, in this species, ontogenetic growth seems influenced by habitat-associated selection as well as by gender and age-specific selection. These differing selection regimes result in ontogenetic shifts in allometry in three of the four groups examined.     

Terpene chemodiversity of relict conifers Picea omorika, Pinus heldreichii, and Pinus peuce, endemic to Balkan

Terpenes are often used as ecological and chemotaxonomic markers of plant species, as well as for estimation of geographic variability. Essential oils of relic and Balkan endemic/subendemic conifers, Picea omorika, Pinus heldreichii, and P. peuce, in central part of Balkan Peninsula (Serbia and Montenegro), on the level of terpene classes and common terpene compounds were investigated. In finding terpene combinations, which could show the best diversity between species and their natural populations, several statistical methods were applied. Apart from the content of different terpene classes (P. omorika has the most abundant O-containing monoterpenes and sesquiterpenes; P. heldreichii and P. peuce have the largest abundance of sesquiterpene and monoterpene hydrocarbons, resp.), the species are clearly separated according to terpene profile with 22 common compounds. But, divergences in their populations were established only in combination of several compounds (specific for each species), and they were found to be the results of geomorphologic, climatic, and genetic factors. We found similarities between investigated species and some taxa from literature with respect to terpene composition, possibly due to hybridization and phylogenetic relations. Obtained results are also important regarding to chemotaxonomy, biogeography, phylogeny, and evolution of these taxa.     

Volatile terpenes – mediators of plant-to-plant communication

Plants interact with other organisms employing volatile organic compounds (VOCs). The largest group of plant-released VOCs are terpenes, comprised of isoprene, monoterpenes, and sesquiterpenes. Mono- and sesquiterpenes are well-known communication compounds in plant–insect interactions, whereas the smallest, most commonly emitted terpene, isoprene, is rather assigned a function in combating abiotic stresses. Recently, it has become evident that different volatile terpenes also act as plant-to-plant signaling cues. Upon being perceived, specific volatile terpenes can sensitize distinct signaling pathways in receiver plant cells, which in turn trigger plant innate immune responses. This vastly extends the range of action of volatile terpenes, which not only protect plants from various biotic and abiotic stresses, but also convey information about environmental constraints within and between plants. As a result, plant–insect and plant–pathogen interactions, which are believed to influence each other through phytohormone crosstalk, are likely equally sensitive to reciprocal regulation via volatile terpene cues. Here, we review the current knowledge of terpenes as volatile semiochemicals and discuss why and how volatile terpenes make good signaling cues. We discuss how volatile terpenes may be perceived by plants, what are possible downstream signaling events in receiver plants, and how responses to different terpene cues might interact to orchestrate the net plant response to multiple stresses. Finally, we discuss how the signal can be further transmitted to the community level leading to a mutually beneficial community-scale response or distinct signaling with near kin.     

The Eucalyptus terpene synthase gene family

Background

Terpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts.

Results

The genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissues and functionally characterized five TPS genes from E. grandis. Compared to other sequenced plant genomes, Eucalyptus grandis has the largest number of putative functional TPS genes of any sequenced plant. We discovered 113 and 106 putative functional TPS genes in E. grandis and E. globulus, respectively. All but one TPS from E. grandis were expressed in at least one of seven plant tissues examined. Genomic clusters of up to 20 genes were identified. Many TPS are expressed in tissues other than leaves which invites a re-evaluation of the function of terpenes in Eucalyptus.

Conclusions

Our data indicate that terpenes in Eucalyptus may play a wider role in biotic and abiotic interactions than previously thought. Tissue specific expression is common and the possibility of stress induction needs further investigation. Phylogenetic comparison of the two investigated Eucalyptus species gives insight about recent evolution of different clades within the TPS gene family. While the majority of TPS genes occur in orthologous pairs some clades show evidence of recent gene duplication, as well as loss of function.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1598-x) contains supplementary material, which is available to authorized users.     

Terpene emission in tissue culture

Tissue cultured plants’ vessel headspace is subject to changes during subculture, and the analysis of its variation offers a non-destructive approach for monitoring plant physiology. Among the volatile organic compounds (VOCs) that can be released by plants and be potentially recovered in the airspace of plant tissue cultures, terpenes are very important since they can offer a snapshot of the physiological status of the plant under in vitro cultivation. Terpenes are synthesized from carbon directly shunted from the photosynthetic carbon fixation cycle and their emission is under genetic and environmental control. The experiments described in this paper propose the evaluation of the plant terpene profile in the culture’s headspace as an early indicator of plant stress through the characterization of plant terpene production. Monitoring of terpene emission as a plant response to mechanical stress such as plant wounding showed an increased isoprene and monoterpene emission rates in the first hour after cutting. The comparison of headspace composition of cultures of two fruit rootstocks, Colt and GF677, showed the former having higher emissions of isoprene, α-pinene and limonene than the latter. A decreasing emission trend was observed during subculture, apparently as a result of culture aging. The in vitro headspace analysis of different myrtle (Myrtus communis L.) clones showed a specific and distinctive terpene emission profile. VOC monitoring of in vitro culture headspace is discussed as a non-destructive approach useful for its relation with major physiological activities of culture and for the determination of the potential production of terpenes.     

The influence of genetics,defensive chemistry and the fungal microbiome on disease outcome in whitebark pine trees

The invasive fungal pathogen Cronartium ribicola infects and kills whitebark pine (Pinus albicaulis) throughout western North America. Whitebark pine has been proposed for listing under the Endangered Species Act in the USA, and the loss of this species is predicted to have severe impacts on ecosystem composition and function in high‐elevation forests. Numerous fungal endophytes live inside whitebark pine tissues and may influence the severity of C. ribicola infection, either directly by inhibition of pathogen growth or indirectly by the induction of chemical defensive pathways in the tree. Terpenes, a form of chemical defence in pine trees, can also influence disease. In this study, we characterized fungal endophyte communities in whitebark pine seedlings before and after experimental inoculation with C. ribicola, monitored disease progression and compared fungal community composition in susceptible vs. resistant seedlings in a common garden. We analysed the terpene composition of these same seedlings. Seed family identity or maternal genetics influenced both terpenes and endophyte communities. Terpene and endophyte composition correlated with disease severity, and terpene concentrations differed in resistant vs. susceptible seedlings. These results suggest that the resistance to C. ribicola observed in natural whitebark pine populations is caused by the combined effects of genetics, endophytes and terpenes within needle tissue, in which initial interactions between microbes and hosts take place. Tree genotype, terpene and microbiome combinations associated with healthy trees could help to predict or reduce disease severity and improve outcomes of future tree breeding programmes.     

Induction of terpene biosynthesis in dinoflagellate symbionts of Caribbean gorgonians

This report describes a series of experiments designed to determine if terpene biosynthesis is inducible in two families of marine terpenes, pseudopterosins from the gorgonian coral Pseudopterogorgia elisabethae and fuscol from Eunicea fusca. Since we have recently shown that terpene biosynthesis is not under the control of the invertebrate host, but rather occurs within a dinoflagellate preparation, we examined the terpene content of the dinoflagellate symbiont following a decrease in UV/vis radiation as well as in response to the addition of methyl jasmonate, salicylic acid and gibberellic acid. We demonstrated that pseudopterosin and fuscol biosynthesis can be markedly increased through the addition of the plant bioactive substances. We also demonstrated that, while the terpene content of P. elisabethae increases in response to decreased UV/vis light, this is due primarily to an increase in the concentration of the dinoflagellate rather than simply an induction of terpene biosynthesis.