A claim for a 'next generation' of multisite range-wide forest genetic trials built on the legacy of ecological genetics to anticipate responses to climate.

Realizamos un comentario sobre el trabajo de revisión presentado por Leites & Benito-Garzón (2023) en este número sobre el valor de las redes de ensayos genéticos forestales multi-sitio para anticipar la distribución y las respuestas de las especies forestales a la actual crisis climática. La persistencia de las masas forestales y de los servicios ecosistémicos que proporcionan necesita de un esfuerzo coordinado sin precedentes de la comunidad científica para comprender, predecir y mitigar los impactos de la crisis climática sobre los bosques templados y boreales. Para ello se necesita información precisa sobre la variación genética, plasticidad fenotípica y la variación genética en plasticidad de las especies forestales. Hacemos una llamada de atención sobre la necesidad urgente de establecer una nueva generación de ensayos genéticos forestales de procedencias replicados multisitio a lo largo del rango de distribución de las especies de interés, y enfocado en la resiliencia al cambio climático. Como científicos debemos facilitar las respuestas evolutivas a los tan rápidos que están viviendo las especies forestales. Debemos ser determinantes en proporcionar información de primera clase a los actores políticos y administradores para desarrollar una gestión forestal basada en la evidencia científica.

Drivers of population differentiation in phenotypic plasticity in a temperate conifer: A 27-year study.

Phenotypic plasticity is a main mechanism for organisms to cope with changing environments and broaden their ecological range. Plasticity is genetically based and can evolve under natural selection, such that populations within a species show distinct phenotypic responses to the environment if evolved under different conditions. Understanding how intraspecific variation in phenotypic plasticity arises is critical to assess potential adaptation to ongoing climate change. Theory predicts that plasticity is favored in more favorable but variable environments. Yet, many theoretical predictions about benefits, costs, and selection on plasticity remain untested. To test these predictions, we took advantage of three genetic trials in the northern Rocky Mountains, USA, which assessed 23 closely located Pinus ponderosa populations over 27 years. Mean environmental conditions and their spatial patterns of variation at the seed source populations were characterized based on six basic climate parameters. Despite the small area of origin, there was significant genetic variation in phenotypic plasticity for tree growth among populations. We found a significant negative correlation between phenotypic plasticity and the patch size of environmental heterogeneity at the seed source populations, but not with total environmental spatial variance. These results show that populations exposed to high microhabitat heterogeneity have evolved higher phenotypic plasticity and that the trigger was the grain rather than the total magnitude of spatial heterogeneity. Contrary to theoretical predictions, we also found a positive relationship between population plasticity and summer drought at the seed source, indicating that drought can act as a trigger of plasticity. Finally, we found a negative correlation between the quantitative genetic variance within populations and their phenotypic plasticity, suggesting compensatory adaptive mechanisms for the lack of genetic diversity. These results improve our understanding of the microevolutionary drivers of phenotypic plasticity, a critical process for resilience of long-lived species under climate change, and support decision-making in tree genetic improvement programs and seed transfer strategies.

Phenotypic, epigenetic, and fitness diversity within plant genotypes.

Plant plastic responses to environmental variation, at scales smaller than the individual plant size, promote phenotypic and epigenetic diversity among repeated structures within genotypes. Different epigenetic marks in the somatic line can translate to the germline and seeds, generating a fitness patchwork in the progeny with unexplored effects on plant evolutionary dynamics.

Phenotypic plasticity in plant defense across life stages: Inducibility, transgenerational induction, and transgenerational priming in wild radish.

As they develop, many plants deploy shifts in antiherbivore defense allocation due to changing costs and benefits of their defensive traits. Plant defenses are known to be primed or directly induced by herbivore damage within generations and across generations by long-lasting epigenetic mechanisms. However, little is known about the differences between life stages of epigenetically inducible defensive traits across generations. To help fill this knowledge gap, we conducted a multigenerational experiment to determine whether defense induction in wild radish plants was reflected in chromatin modifications (DNA methylation); we then examined differences between seedlings and reproductive plants in current and transgenerational plasticity in chemical (glucosinolates) and physical (trichomes) defenses in this species. Herbivory triggered genome methylation both in targeted plants and their offspring. Within one generation, both defenses were highly inducible at the seedling stage, but only chemical defenses were inducible in reproductive plants. Across generations, herbivory experienced by mother plants caused strong direct induction of physical defenses in their progeny, with effects lasting from seedling to reproductive stages. For chemical defenses, however, this transgenerational induction was evident only in adults. Transgenerational priming was observed in physical and chemical defenses, particularly in adult plants. Our results show that transgenerational plasticity in plant defenses in response to herbivore offense differs for physical and chemical defense and changes across plant life stages.

Direct and correlated responses to artificial selection for growth and water-use efficiency in a Mediterranean pine.

PREMISE: Persistence of tree populations in the face of global change relies on their capacity to respond to biotic and abiotic stressors through plastic or adaptive changes. Genetic adaptation will depend on the additive genetic variation within populations and the heritability of traits related to stress tolerance. Because traits can be genetically linked, selective pressure acting on one trait may lead to correlated responses in other traits. METHODS: To test direct and correlated responses to selection for growth and drought tolerance in Pinus halepensis, we selected trees in a parental population for higher growth and greater water-use efficiency (WUE) and compared their offspring with the offspring of random trees from the parental population in two contrasting common gardens. We estimated direct responses to selection for growth and WUE and correlated responses for growth and tolerance to abiotic and biotic stressors. RESULTS: We found a strong response to selection and high realized heritability for WUE, but no response to selection for growth. Correlated responses to selection in other life-history traits were not significant, except for concentration of some chemical defenses, which was greater in the offspring of mother trees selected for growth than in the offspring of unselected control trees. CONCLUSIONS: The empirical evidence of direct responses to selection for high WUE suggests that P. halepensis has the potential to evolve in response to increasing drought stress. Contrary to expectations, the results are not conclusive of a potential negative impact of WUE and growth selection on other key life-history traits.

Insect defoliation is linked to a decrease in soil ectomycorrhizal biomass and shifts in needle endophytic communities.

Insect outbreaks of increasing frequency and severity in forests are predicted due to climate change. Insect herbivory is known to promote physiological changes in forest trees. However, little is known about whether these plant phenotypic adjustments have cascading effects on tree microbial symbionts such as fungi in roots and foliage. We studied the impact of defoliation by the pine processionary moth in two infested Pinus nigra forests through a multilevel sampling of defoliated and non-defoliated trees. We measured tree growth, nutritional status and carbon allocation to chemical defenses. Simultaneously, we analysed the putative impact of defoliation on the needle endophytes and on the soil fungal communities. Higher concentrations of chemical defenses were found in defoliated trees, likely as a response to defoliation; however, no differences in non-structural carbohydrate reserves were found. In parallel to the reductions in tree growth and changes in chemical defenses, we observed shifts in the composition of needle endophytic and soil fungal communities in defoliated trees. Defoliated trees consistently corresponded with a lower biomass of ectomycorrhizal fungi in both sites, and a higher alpha diversity and greater relative abundance of belowground saprotrophs and pathogens. However, ectomycorrhizal alpha diversity was similar between non-defoliated and defoliated trees. Specific needle endophytes in old needles were strongly associated with non-defoliated trees. The potential role of these endophytic fungi in pine resistance should be further investigated. Our study suggests that lower biomass of ectomycorrhizal fungi in defoliated trees might slow down tree recovery since fungal shifts might affect tree-mycorrhizal feedbacks and can potentially influence carbon and nitrogen cycling in forest soils.

Climate drives intraspecific differentiation in the expression of growth-defence trade-offs in a long-lived pine species.

Intraspecific variation in plant defences is expected to be the result of adaptive and plastic responses to environmental conditions, where trade-offs between growth and defences are thought to play a key role shaping phenotypic patterns in defensive investment. Axial resin ducts are costly defensive structures that remain imprinted in the tree rings of conifers, therefore being a valuable proxy of defensive investment along the trees' lifespan. We aimed to disentangle climate-driven adaptive clines and plastic responses to both spatial and temporal environmental variation in resin duct production, and to explore growth-defence trade-offs. To that aim, we applied dendrochronological procedures to quantify annual growth and resin duct production during a 31-year-period in a Mediterranean pine species, including trees from nine populations planted in two common gardens. Both genetic factors and plastic responses modulated annual resin duct production. However, we found no evidence of adaptive clines with climate gradients driving population differentiation. Our results revealed a marked physiological trade-off between growth and defences, where the slope of the trade-off was genetically variable and associated with climatic gradients. Our results help to enlighten the evolutionary patterns and genetic basis of defensive allocation within species, particularly revealing a key role of growth-defence trade-offs.

Resin ducts as resistance traits in conifers: linking dendrochronology and resin-based defences.

Conifers have evolved different chemical and anatomical defences against a wide range of antagonists. Resin ducts produce, store and translocate oleoresin, a complex terpenoid mixture that acts as both a physical and a chemical defence. Although resin duct characteristics (e.g., number, density, area) have been positively related to biotic resistance in several conifer species, the literature reporting this association remains inconclusive. Axial resin ducts recorded in annual growth rings are an archive of annual defensive investment in trees. This whole-life record of defence investment can be analysed using standard dendrochronological procedures, which allows us to assess interannual variability and the effect of understudied drivers of phenotypic variation on resin-based defences. Understanding the sources of phenotypic variation in defences, such as genetic differentiation and environmental plasticity, is essential for assessing the adaptive potential of forest tree populations to resist pests under climate change. Here, we reviewed the evidence supporting the importance of resin ducts in conifer resistance, and summarized current knowledge about the sources of variation in resin duct production. We propose a standardized methodology to measure resin duct production by means of dendrochronological procedures. This approach will illuminate the roles of resin ducts in tree defence across species, while helping to fill pivotal knowledge gaps in plant defence theory, and leading to a robust understanding of the patterns of variation in resin-based defences throughout the tree's lifespan.

Resin acids as inducible chemical defences of pine seedlings against chewing insects.

Inducibility of defences in response to biotic stimuli is considered an important trait in plant resistance. In conifers, previous research has mostly focused on the inducibility of the volatile fraction of the oleoresin (mono- and sesquiterpenes), leaving the inducibility of the non-volatile resin acids largely unexplored, particularly in response to real herbivory. Here we investigated the differences in the inducibility of resin acids in two pine species, one native from Europe (Pinus pinaster Ait.) and another from North America (Pinus radiata D. Don), in response to wounding by two European insects: a bark chewer, the pine weevil (Hylobius abietis L.), and a defoliator, the pine processionary caterpillar (Thaumetopoea pityocampa Schiff.). We quantified the constitutive (control) and induced concentrations of resin acids in the stem and needles of both pine species by gas chromatography techniques. Both pine species strongly increased the concentration of resin acids in the stem after pine weevil feeding, although the response was greater in P. pinaster than in P. radiata. However, systemic defensive responses in the needles were negligible in both pine species after pine weevil feeding in the stem. On the other hand, P. radiata locally reduced the resin acid concentration in the needles after pine caterpillar feeding, whereas in P. pinaster resin acid concentration was apparently unaffected. Nevertheless, systemic induction of resin acids was only observed in the stem of P. pinaster in response to pine caterpillar feeding. In summary, pine induced responses were found highly compartmentalized, and specific to herbivore identity. Particularly, plant defence suppression mechanisms by the pine caterpillar, and ontogenetic factors might be potentially affecting the induced response of resin acids in both pine species.

Neutral and Climate-Driven Adaptive Processes Contribute to Explain Population Variation in Resin Duct Traits in a Mediterranean Pine Species.

Resin ducts are important anatomical defensive traits related to biotic resistance in conifers. Previous studies have reported intraspecific genetic variation in resin duct characteristics. However, little is currently known about the micro-evolutionary patterns and adaptive value of these defensive structures. Here, we quantified inter-population genetic variation in resin duct features and their inducibility in Pinus pinaster and assessed whether such variation was associated with climate gradients. To that end, we characterized the resin duct system of 2-year-old saplings from 10 populations across the species' distribution range. We measured axial resin duct features (density, mean size, and percentage conductive area of resin ducts) and their inducibility in response to methyl jasmonate. Genotyping of single nucleotide polymorphisms allowed to account for the population genetic structure in our models in order to avoid spurious correlations between resin duct characteristics and climate. We found large inter-population variation in resin duct density and conductive area, but not in their inducibility. Our results suggest that population variation in the percentage conductive area of resin ducts likely arise from adaptation to local climate conditions. This study highlights the adaptive relevance of resin ducts and helps to shed light on the micro-evolutionary patterns of resin-based defenses in conifers.

Inducibility of Plant Secondary Metabolites in the Stem Predicts Genetic Variation in Resistance Against a Key Insect Herbivore in Maritime Pine.

Resistance to herbivores and pathogens is considered a key plant trait with strong adaptive value in trees, usually involving high concentrations of a diverse array of plant secondary metabolites (PSM). Intraspecific genetic variation and plasticity of PSM are widely known. However, their ecology and evolution are unclear, and even the implication of PSM as traits that provide direct effective resistance against herbivores is currently questioned. We used control and methyl jasmonate (MJ) induced clonal copies of genotypes within families from ten populations of the main distribution range of maritime pine to exhaustively characterize the constitutive and induced profile and concentration of PSM in the stem phloem, and to measure insect herbivory damage as a proxy of resistance. Then, we explored whether genetic variation in resistance to herbivory may be predicted by the constitutive concentration of PSM, and the role of its inducibility to predict the increase in resistance once the plant is induced. We found large and structured genetic variation among populations but not between families within populations in resistance to herbivory. The MJ-induction treatment strongly increased resistance to the weevil in the species, and the genetic variation in the inducibility of resistance was significantly structured among populations, with greater inducibility in the Atlantic populations. Genetic variation in resistance was largely explained by the multivariate concentration and profile of PSM at the genotypic level, rather than by bivariate correlations with individual PSM, after accounting for genetic relatedness among genotypes. While the constitutive concentration of the PSM blend did not show a clear pattern of resistance to herbivory, specific changes in the chemical profile and the increase in concentration of the PSM blend after MJ induction were related to increased resistance. To date, this is the first example of a comprehensive and rigorous approach in which inducibility of PSM in trees and its implication in resistance was analyzed excluding spurious associations due to genetic relatedness, often overlooked in intraspecific studies. Here we provide evidences that multivariate analyses of PSM, rather than bivariate correlations, provide more realistic information about the potentially causal relationships between PSM and resistance to herbivory in pine trees.

Effect of Light Availability on the Interaction between Maritime Pine and the Pine Weevil: Light Drives Insect Feeding Behavior But Also the Defensive Capabilities of the Host.

Light is a major environmental factor that may determine the interaction between plants and herbivores in several ways, including top-down effects through changes in herbivore behavior and bottom-up effects mediated by alterations of plant physiology. Here we explored the relative contribution of these two regulation processes to the outcome of the interaction of pine trees with a major forest pest, the pine weevil (Hylobius abietis). We studied to what extent light availability influence insect feeding behavior and/or the ability of pines to produce induced defenses in response to herbivory. For this purpose, 3-year old Pinus pinaster plants from three contrasting populations were subjected to 6 days of experimental herbivory by the pine weevil under two levels of light availability (complete darkness or natural sunlight) independently applied to the plant and to the insect in a fully factorial design. Light availability strongly affected the pine weevil feeding behavior. The pine weevil fed more and caused larger feeding scars in darkness than under natural sunlight. Besides, under the more intense levels of weevil damage (i.e., those registered with insects in darkness), light availability also affected the pine's ability to respond to insect feeding by producing induced resin defenses. These results were consistent across the three studied populations despite they differed in weevil susceptibility and inducibility of defenses. Morocco was the most damaged population and the one that induced more defensive compounds. Overall, results indicate that light availability modulates the outcome of the pine-weevil interactions through both bottom-up and top-down regulation mechanisms.

Defensive Traits in Young Pine Trees Cluster into Two Divergent Syndromes Related to Early Growth Rate.

The combination of defensive traits leads to the evolution of 'plant defense syndromes' which should provide better protection against herbivores than individual traits on their own. Defense syndromes can be generally driven by plant phylogeny and/or biotic and abiotic factors. However, we lack a solid understanding of (i) the relative importance of shared evolution vs. convergence due to similar ecological conditions and (ii) the role of induced defense strategies in shaping defense syndromes. We investigate the relative roles of evolutionary and ecological factors shaping the deployment of pine defense syndromes including multiple constitutive and induced chemical defense traits. We performed a greenhouse experiment with seedlings of eighteen species of Pinaceae family, and measured plant growth rate, constitutive chemical defenses and their inducibility. Plant growth rate, but not phylogenetic relatedness, determined the deployment of two divergent syndromes. Slow-growing pine species living in harsh environments where tissue replacement is costly allocated more to constitutive defenses (energetically more costly to produce than induced). In contrast, fast-growing species living in resource-rich habitats had greater inducibility of their defenses, consistent with the theory of constitutive-induced defense trade-offs. This study contributes to a better understanding of evolutionary and ecological factors driving the deployment of defense syndromes.

Susceptibility to the pinewood nematode (PWN) of four pine species involved in potential range expansion across Europe.

The pine wilt disease (PWD), caused by the pinewood nematode (PWN) Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, is one of the most serious threats to pine forests worldwide. Here we studied several components of susceptibility to PWN infection in a model group of pine species widely distributed in Europe (Pinus pinaster Ait., P. pinea L., P. sylvestris L. and P. radiata D. Don), specifically concerning anatomical and chemical traits putatively related to nematode resistance, whole-plant nematode population after experimental inoculation, and several biochemical and physiological traits indicative of plant performance, damage and defensive responses 60 days post inoculation (dpi) in 3-year-old plants. Pinus pinaster was the most susceptible species to PWN colonization, with a 13-fold increase in nematode population size following inoculation, showing up to 35-fold more nematodes than the other species. Pinus pinea was the most resistant species, with an extremely reduced nematode population 60 dpi. Axial resin canals were significantly wider in P. pinaster than in the other species, which may have facilitated nematode dispersal through the stem and contributed to its high susceptibility; nevertheless, this trait does not seem to fully determinate the susceptible character of a species, as P. sylvestris showed similar nematode migration rates to P. pinaster but narrower axial resin canals. Nematode inoculation significantly affected stem water content and polyphenolic concentration, and leaf chlorophyll and lipid peroxidation in all species. In general, P. pinaster and P. sylvestris showed similar chemical responses after infection, whereas P. radiata, which co-exists with the PWN in its native range, showed some degree of tolerance to the nematode. This work provides evidence that the complex interactions between B. xylophilus and its hosts are species-specific, with P. pinaster showing a strong susceptibility to the pathogen, P. pinea being the most tolerant species, and P. sylvestris and P. radiata having a moderate susceptibility, apparently through distinct coping mechanisms.

Correlated genetic effects on reproduction define a domestication syndrome in a forest tree.

Compared to natural selection, domestication implies a dramatic change in traits linked to fitness. A number of traits conferring fitness in the wild might be detrimental under domestication, and domesticated species typically differ from their ancestors in a set of traits known as the domestication syndrome. Specifically, trade-offs between growth and reproduction are well established across the tree of life. According to allocation theory, selection for growth rate is expected to indirectly alter life-history reproductive traits, diverting resources from reproduction to growth. Here we tested this hypothesis by examining the genetic change and correlated responses of reproductive traits as a result of selection for timber yield in the tree Pinus pinaster. Phenotypic selection was carried out in a natural population, and progenies from selected trees were compared with those of control trees in a common garden experiment. According to expectations, we detected a genetic change in important life-history traits due to selection. Specifically, threshold sizes for reproduction were much higher and reproductive investment relative to size significantly lower in the selected progenies just after a single artificial selection event. Our study helps to define the domestication syndrome in exploited forest trees and shows that changes affecting developmental pathways are relevant in domestication processes of long-lived plants.

Differentiation of persistent anatomical defensive structures is costly and determined by nutrient availability and genetic growth-defence constraints.

Conifers exhibit a number of chemical and anatomical mechanisms to defend against pests and pathogens. Theory predicts an increased investment in plant defences under limited nutrient availability, but while this has been demonstrated for chemical defences, it has rarely been shown for anatomical defensive structures. In a long-lived woody plant, we tested the hypothesis that limited nutrient availability may promote an improved differentiation of persistent anatomical defences. We also hypothesized that the costs of differentiation of those long-term anatomical structures may be determined by genetic constraints on early growth potential. Using Pinus pinaster Ait. juveniles, we performed a greenhouse study with 15 half-sib families subjected to experimental manipulation of phosphorus (P) availability and herbivory-related induced responses. When plants were ∼30 cm high, half of the plant material was treated with methyl jasmonate to induce defences, and 2 weeks later plants were harvested and the abundance of resin canals in the cortex and xylem was assessed. Density of constitutive resin canals in the cortex and the total canal system was ∼1.5-fold higher in plants under limited P availability than in fully fertilized plants. Availability of P did not significantly influence the inducibility of resin canal traits. We found negative genetic correlations between plant growth and the density of constitutive canals in the xylem and total canal system, but only under conditions of limited nutrition. These results demonstrate for the first time that differentiation of constitutive anatomical-based defences is affected by P limitation. Moreover, results also evidence the existence of genetic constraints between plant growth and constitutive defensive investment, where lineages with the highest growth potential showed the lowest investment in constitutive resin canals.

Trade-offs between constitutive and induced defences drive geographical and climatic clines in pine chemical defences.

There is increasing evidence that geographic and climatic clines drive the patterns of plant defence allocation and defensive strategies. We quantified early growth rate and both constitutive and inducible chemical defences of 18 Pinaceae species in a common greenhouse environment and assessed their defensive allocation with respect to each species' range across climatic gradients spanning 31° latitude and 2300 m elevation. Constitutive defences traded-off with induced defences, and these defensive strategies were associated with growth rate such that slow-growing species invested more in constitutive defence, whereas fast-growing species invested more in inducible defence. The position of each pine species along this trade-off axis was in turn associated with geography; moving poleward and to higher elevations, growth rate and inducible defences decreased, while constitutive defence increased. These geographic patterns in plant defence were most strongly associated with variation in temperature. Climatic and geographical clines thus act as drivers of defence profiles by mediating the constraints imposed by trade-offs, and this dynamic underlays global patterns of defence allocation.

Environmental maternal effects mediate the resistance of maritime pine to biotic stress.

The resistance to abiotic stress is increasingly recognised as being impacted by maternal effects, given that environmental conditions experienced by parent (mother) trees affect stress tolerance in offspring. We hypothesised that abiotic environmental maternal effects may also mediate the resistance of trees to biotic stress. The influence of maternal environment and maternal genotype and the interaction of these two factors on early resistance of Pinus pinaster half-sibs to the Fusarium circinatum pathogen was studied using 10 mother genotypes clonally replicated in two contrasting environments. Necrosis length of infected seedlings was 16% shorter in seedlings grown from favourable maternal environment seeds than in seedlings grown from unfavourable maternal environment seeds. Damage caused by F. circinatum was mediated by maternal environment and maternal genotype, but not by seed mass. Mechanisms unrelated to seed provisioning, perhaps of epigenetic nature, were probably involved in the transgenerational plasticity of P. pinaster, mediating its resistance to biotic stress. Our findings suggest that the transgenerational resistance of pines due to an abiotic stress may interact with the defensive response of pines to a biotic stress.

Inducibility of chemical defences by two chewing insect herbivores in pine trees is specific to targeted plant tissue, particular herbivore and defensive trait.

There is increasing evidence that plants can react to biotic aggressions with highly specific responses. However, few studies have attempted to jointly investigate whether the induction of plant defences is specific to a targeted plant tissue, plant species, herbivore identity, and defensive trait. Here we studied those factors contributing to the specificity of induced defensive responses in two economically important pine species against two chewing insect pest herbivores. Juvenile trees of Pinus pinaster and P. radiata were exposed to herbivory by two major pest threats, the large pine weevil Hylobius abietis (a bark-feeder) and the pine processionary caterpillar Thaumetopoea pityocampa (a folivore). We quantified in two tissues (stem and needles) the constitutive (control plants) and herbivore-induced concentrations of total polyphenolics, volatile and non-volatile resin, as well as the profile of mono- and sesquiterpenes. Stem chewing by the pine weevil increased concentrations of non-volatile resin, volatile monoterpenes, and (marginally) polyphenolics in stem tissues. Weevil feeding also increased the concentration of non-volatile resin and decreased polyphenolics in the needle tissues. Folivory by the caterpillar had no major effects on needle defensive chemistry, but a strong increase in the concentration of polyphenolics in the stem. Interestingly, we found similar patterns for all these above-reported effects in both pine species. These results offer convincing evidence that induced defences are highly specific and may vary depending on the targeted plant tissue, the insect herbivore causing the damage and the considered defensive compound.