Resin ducts as a retrospective proxy of defensive allocation in a Mediterranean pine speciesadaptive variation and plastic responses

Resumen

Plants have developed a myriad mechanical and chemical defensive mechanisms to minimize the damage caused by the broad and diverse community of antagonist organisms that feed on their tissues. Mechanical defences include those that provide toughness and strength to plant tissues to reduce their palatability, or those that act as a physical or anatomical barrier to avoid the entrance of organisms. Alternatively, chemical defences are plant secondary metabolites that act as toxic or deterrent compounds against invader antagonists. According to the ‘arms race theory’ plant defensive mechanisms have evolved as the result of tight co-evolutionary interactions and counter adaptations between host plants and their herbivores. This theory highlighted the relevance of plant-organism interactions in an evolutionary context and motivated further research to understand the patterns of variation in plant defences. Plant defences are known to vary widely both among and within species. Differences in defensive investment among species are usually associated with geographic patterns in biotic and abiotic factors where adaptation to the local environment is expected to drive macroevolution patterns of plant defences. For instance, allocation to defences is expected to be maximised in environments with high herbivorous pressure where it implies an increase in plant fitness. Resource availability is also known to be a key factor modulating plant-organism interactions and the macroevolution of plant defences, as the cost-benefit ratio of defence production depends largely on resource acquisition. Patterns of defensive allocation within species, including variation among and within populations, are also the result of an intricate network of various factors, including genetic, environmental and ontogenetic among others. Genetic variation within species has been extensively reported for chemical and anatomical defences across a wide range of plant species. In a similar way to variation among species, adaptation to heterogeneous environmental conditions in biotic pressures and/or abiotic conditions across a species distribution range can lead to genetic differentiation of populations within species. Among-population variation in defences can also arise from genetic drift and particular demographic processes, leading to patterns of neutral, non-adaptive, genetic variation. Genetic variation in plant defences can also be found within populations, namely among families or individuals. Gene flow, mutation and migration within a population distribution range might contribute to maintain high levels of additive genetic variation within populations, which ultimately constitute the fuel of natural selection. Environmental factors can also affect defence production contributing to phenotypic variation within species. Because defensive compounds and structures are especially costly to produce, plants must optimally accommodate defensive investment according to their particular environment. Plastic responses of defensive traits include those produced as a physiological reaction to biotic damage, widely known as inducible defences, but also responses to abiotic factors such as light and nutrient availability or climate conditions. Furthermore, plastic responses of defensive traits are also known to vary genetically within species. In other words, there is intraspecific genetic variation in reaction norms to environmental gradients. An extensive body of knowledge have focussed in the last decades in understanding the evolutionary and ecological basis underlying the observed patterns of phenotypic variation in plant defences, conforming what we know today as the plant defence theory. Because defences are costly and resources are limited, plants must regulate defence production according to its cost and benefits on their fitness. Particularly, hypotheses concerning the expression of growth-defence trade-offs modulating diversity patterns in plant defences has been particularly relevant. Those hypotheses mainly predict that species or individuals growing under low resource availability will grow less but invest more in defensive traits. However, studies testing the specific predictions of such hypothesis often show contrasting results. Disentangling adaptive patterns underlying genetic differentiation and plastic responses to environmental variation in plant defences within species becomes crucial to forecast the survival and performance of plant populations in the near future. The expansion of native antagonist organisms and the arrival of new pests caused by globalization together with altered climate conditions can suppose a challenge for the persistence of plant species. Under such scenario, plant populations can migrate, adapt, or acclimate to the new environment. Although migration to new optimal environments can be possible, the speed and intensity of ongoing environmental shifts might difficult range expansions. In those cases, populations must adapt to their new environment by means of natural selection. Furthermore, phenotypic plasticity enhancing plant fitness can buffer the negative impacts of environmental stresses until genetic adaptation can occur, being thus especially relevant from a micro-evolutionary perspective. Plant responses to environmental change will also depend enormously on the magnitude of genotype by environment interactions, this is, the extant genetic variation in plasticity. Given the relevance of both adaptive and plastic responses in the evolutionary outcomes of plant species exposed to environmental change, it is highly desirable to simultaneously assess their effects on traits enhancing plant fitness. Conifers are massive, long-lived sessile organisms that have colonized vast regions worldwide, being exposed to wide spatial and long-term temporal variation in their biotic and abiotic environment. As enormous apparent keystone species, conifers have evolved a particularly sophisticated defensive system to defend against their enemies, which has surely contributed to their colonization success. Conifer defences importantly rely on the synthesis and storage of big amounts of terpenoid rich oleoresin (resin hereafter). Terpenoid resin is accumulated and synthesised in conifers in structures varying in complexity, from simple cells and resin blisters to complex tub-like structures such as resin ducts in Pinaceae trees. Particularly, resin ducts are permanent and costly structures, and their production is assumed to be positively associated with resistance to a number of insects and pathogens. Importantly, axial resin ducts placed longitudinally along the stem, roots and branches of conifers, are imprinted in the xylem tree rings, allowing their retrospective quantification through dendrochronological procedures. This approach allows to build annually resolved series of defensive investment, thereby accounting for the temporal variation in conifer defences along their lifespan. The retrospective resin duct analysis enables to explore the effects of particular climate conditions driving interannual variation in defence production. Moreover, dendrochronological procedures applied to mature trees in common gardens can help to disentangle the genetic basis of defence production. In this Doctoral Thesis, genetic and environmental factors driving the expression of resin-based anatomical defences were explored along the lifespan of trees using maritime pine (Pinus pinaster Ait.) as a model Mediterranean pine species and resin duct characteristics as a proxy of defensive investment. P. pinaster is a conifer tree of high ecological and economic value distributed from southern Europe to Northern Africa, inhabiting wide regions that vary strongly in their environmental conditions. As other Mediterranean pine species, P. pinaster has today a fragmented distribution that is the result of a singular demographic history of postglacial migration routes from specific glacial refugia. Accordingly, populations of this species are strongly differentiated for molecular and functional traits reflecting patterns of neutral variation and adaptive processes. These characteristics make P. pinaster a valuable species for studying intraspecific genetic variation and microevolutionary patterns in defensive allocation. More specifically, the objectives of this thesis were (i) to assess the relevance of higher resin duct production on conifer resistance by testing whether resistant trees invest more in resin duct production than susceptible trees, (ii) to explore intraspecific genetic variation (among populations) in constitutive (always present) and induced (produced in response to biotic damage) resin ducts and to determine the existence of adaptive clines associated with climate gradients driving genetic differentiation among populations of Pinus pinaster, (iii) to determine the existence of trade-offs between growth (annual radial growth) and defences (annual production of resin ducts) and whether the expression of such trade-offs are variable among populations of Pinus pinaster and associated with climate gradients at the origin of populations, and finally, (iv) to explore phenotypic plasticity (both temporal and spatial) in resin duct production, determining which particular climate variables might affect the production of this anatomical trait, and to assess whether plastic responses are variable among populations of Pinus pinaster (genetic variation in plasticity). To meet these objectives, different approaches where used, from a systematic review to manipulative glasshouse experiments with young saplings and phenotyping of long-term provenance trials with mature reproductive trees. The different chapters conforming this thesis covered, jointly or individually, the above mentioned objectives. The first chapter was conceived as a systematic review that synthesises the existing knowledge on sources of environmental and genetic variation in resin duct traits and their association with enhanced biotic resistance across conifer species. The second chapter explores among population variation in the constitutive production of resin ducts and their inducibility, and deepens in the existence of adaptive clines driving such variation in young P. pinaster saplings. The third chapter uses retrospective resin duct analysis in reproductive P. pinaster trees to explore the interactive effects of genetic variation among populations and plastic responses to spatial environmental variation in anatomical defences. Importantly, the third chapter also assesses the expression of growth-defence trade-offs and explores adaptive clines driving genetic variation in their expression. Finally, the fourth chapter uses annual series of resin duct production to explore how interannual variation in climate conditions drive year-to-year production of anatomical defences and whether such responses to temporal variation in climate conditions differ among populations. The results arising from this comprehensive evaluation of the source of variation in pine anatomical defences contribute to fill current knowledge gaps in plant defence theory. The first objective of this thesis was covered in the chapter 1, where data of resin duct production in resistant and susceptible trees where collected from studies testing whether resin ducts characteristics were associated with enhanced biotic resistance. Resin duct metrics considered included their size, density and number in the bark or xylem of different conifer species. A meta-analysis was applied to the collected data to test for a significant mean effect size of resin duct production on resistant trees. Results from the meta-analysis revealed that resistant trees allocate more to resin duct production than susceptible trees, especially when considering the size of resin ducts. However, there was an enormous heterogeneity across studies, revealing the need for adding more studies testing such association and for a unified methodology. In addition to the meta-analysis, the first chapter comprises an exhaustive bibliographic review to synthesise the current knowledge on the genetic and environmental sources of variation in resin duct characteristics, and thus constitutes the theoretical framework of this thesis. The second objective of this thesis was covered in the chapter II, where genetic variation among populations of P. pinaster in constitutive resin ducts and their inducibility was explored using clonally replicated young individuals. Moreover, adaptive clines following environmental gradient were explored by assessing the association between genetic differentiation among populations and climate at the origin of populations. To that end, the resin duct system of 2-years-old saplings from 10 populations across the species’ distribution range was characterised. Axial resin duct characteristics (density, mean size and percentage conductive area of resin ducts) and their inducibility in response to methyl jasmonate were measured in separate clonal copies. Genotyping of single nucleotide polymorphisms (SNPs) allowed to account for the population genetic structure to avoid spurious correlations between resin duct characteristics and climate. Results revealed large inter-population variation in resin duct density and conductive area, but not in their inducibility. Results obtained in this chapter suggest that population differentiation in the percentage conductive area of resin ducts likely arise from adaptation to local climate conditions, highlighting the adaptive relevance of resin ducts and helping to shed light on the micro-evolutionary patterns of resin-based defences in conifers. The chapter III covers simultaneously the second, third and fourth objectives of this thesis. Genetic variation among populations and plastic responses to spatial environmental heterogeneity (and their interaction) in annual resin duct production were assessed in reproductive P. pinaster trees planted in two common gardens differing in soil characteristics and climate conditions. A total of nine populations were included in the study. A negative association between growth and defences was explored, and genetic variation in both annual resin duct production and in the yearly expression of growth-defence trade-offs were tested for association with climate at the origin of populations. With that purpose, radial growth and resin duct number and density were retrospectively quantified during a 31-year-period in the annual growth rings of trees. Dendrochronological methods where therefore applied to capture interannual variation in resin duct production. Resin duct production was influenced by the species genetic background (genetic variation among populations) and by plastic responses to both interannual environmental variation and local site conditions. Population variation in resin ducts was not associated with climate at the origin. Resin duct density was negatively correlated with the basal area increment across years, revealing a marked physiological growth-defence trade-off. Interestingly, the slope of such negative association was not homogeneous but rather highly variable among populations following climatic clines. Atlantic populations expressed a more pronounced growth-defence trade-off than Mediterranean populations. Results from this chapter reveal a key role of growth-defence trade-offs and suggest and adaptive origin to local climate conditions driving such constraints. We emphasize the usefulness of dendrochronological techniques to anatomically quantify resin-based defences in conifers and assess growth-defence constraints. We aim to stress how this approach will help to fill pivotal knowledge gaps in plant defence research. The chapter IV of this thesis deepens in the patterns of interannual variation observed in the previous chapter, and particularly explores whether interannual climate variation drives year-to-year production of resin ducts, that is, plasticity to temporal environmental variation. Moreover, genetic variation in plastic responses to climate conditions was tested among populations. With that purpose, annual series of standardized resin duct production from data obtained in the previous chapter were used, and its association with climate variables of temperature and precipitation at annual resolution was explored. Results revealed large temporal plasticity in annual resin canal density. Climate variables had a significant effect on allocation to resin ducts. However, climatic effects on resin duct production explained a low proportion of the total interannual variation. Furthermore, despite large variation among populations in year-to year plasticity of anatomical defences, results revealed that climatic responses were homogeneous among populations, with the exception of climate variables related to water availability, for which we found significant genetic variation on plasticity. Results from this chapter contribute to understand plastic responses to climate in resin-based anatomical defences in long-lived conifer species and help to explain patterns of variation in plant defences, useful knowledge to forecast future responses of forest tree populations to global change. All together, the results obtained in this thesis revealed that defensive production in P. pinaster is the result of an intricate network of genetic determinants and environmental modulators. These results contribute to explain microevolutionary patterns and ecological processes modulating intraspecific variation in defensive traits in conifer trees. More specifically, aspects such as the genetic basis and evolutionary relevance of the relative allocation of resources to defences, as well as their climatic modulation, will help to shed light on unanswered questions in plant defence theory. This knowledge can be included in prediction models aiming to forecast the defensive state of populations under global change scenarios. This doctoral thesis manifests the suitability of dendrochronological methods to quantify interannual variation in defensive investment in long-lived species. This approach allows for a robust assessment of defensive production, as it accounts for a representative long time period through the lifespan of trees. Further research should deepen in the exploration of the sources of variation driving defensive investment in this and other Mediterranean species. The generated knowledge will allow to characterise the forest genetic resources, therefore helping to implement adaptive management plans and conservation programs to palliate the negative impacts of global change on Mediterranean ecosystems.