Cork oak (Quercus suber) is an ecologically and economically important Western Mediterranean tree species in severe risk of decline due to aggravated tree mortality and lack of natural regeneration. In Q. suber, two distinct chloroplast lineages, one of them of trans-specific origin, occur in sympatry in the western half of its species distribution. We hypothesize that selection may drive the maintenance of the two lineages and investigate this hypothesis by sequencing chloroplast genomes of 259 Q. suber samples across 24 locations. Protein-coding chloroplast genes were scanned for selection signal using different codon-based methods. Selection signal was found at different sites and genes, and polymorphism in selected sites was shown to segregate between the two chloroplast lineages. We postulate that trans-specific chloroplast diversity in Q. suber is preserved by balancing selection, rather than resulting exclusively from ongoing introgression. These results correspond to an unusual case of balancing selection on whole plastid genomes in a long-lived woody plant species and have implications on conservation and management practices for Q. suber, which may benefit from taking into account genetic variation in plastid genomes as a possible source of increased adaptive potential for the species.

Several regulators of phellem/cork formation have been identified in recent years, using mainly transcriptomic approaches. However, this developmental process, showing parallels to the functioning of vascular cambium, remains poorly understood. The cork oak tree (Quercus suber L.) exhibits a remarkable ability to form a traumatic phellogen after debarking, enabling sustainable cork production. We aimed at uncovering post-transcriptional mechanisms controlled by miRNAs, specifically involved in regulating phellogen functioning and phellem differentiation in cork oak. To achieve this, we conducted a comparative analysis of the small RNA transcriptome between differentiating phellem and xylem, both originating from secondary meristems (phellogen and vascular cambium). In addition to identifying miRNAs exclusive to phellogen/phellem tissues, we discovered 246 differentially expressed miRNAs between the two tissues, of which 74 are conserved. The most abundant miRNA families found in phellem tissues were MIR165/166, MIR167, MIR168 and MIR390. By analysing miRNA predicted targets and their expression in the same tissues, many of the differentially expressed miRNAs were found associated with sequence-specific DNA binding functions. Within these, transcription factor families HD-ZIP III, WRKY, NAC and MYB were highlighted as key in phellem differentiation. Furthermore, hormone-mediated signalling pathways, particularly involving auxin, appeared as an enriched biological process, as several ARF transcripts, among other auxin signalling genes like IAA11, ARF18 and ARF19, were identified as putative targets of conserved or novel miRNAs. Overall, our results provide a comprehensive overview of the miRNA landscape during cork formation, providing valuable knowledge for further functional studies and potential practical applications in forest management.

Stem diameter growth in living trees refers to the invariably increase in dimension over a given period. However, reversible stem diameter decrease could occur, related to water movement in the vessels within the wood (on a daily basis) and to tree water deficit and depletion of stored water (on a seasonal basis). Recently, the perennial decrement in stem diameter size has been related to a tree decline pathway, and eventually resulting in tree death. In this study, we analysed stem diameter intra-annual growth dynamics of holm oak (Quercus rotundifolia Lam.) trees for two full growing seasons in distinct woodlands, Laborela and Aldeia dos Fernandes, in southern Portugal (Ourique district). Our focus was to assess stem diameter growth patterns and trends in holm oak trees in endangered woodlands with high tree mortality and to question if perennial decrement. Holm oaks in Laborela were much more sensitive in their stem diameter variations than in Aldeia dos Fernandes and, on average, their stem diameter decreased continuously by 1.0 mm along the 2-year study period, with a slightly higher annual decrease in the first study year (0.6 mm.yr−1). In addition, on average, trees had a higher decrease in stem sectional area of 5.8 cm2 in Laborela against a decrease of 3.7 cm2 in Aldeia dos Fernandes, where the stem diameter of holm oaks only decreased in the first study year (0.1 mm.yr−1). In each study area, the repeated- measures ANOVA showed that tree size effect influenced the stem diameter variations in contrast to tree crown defoliation. Trees were, on average, relatively smaller in Laborela, with DBH = 35.5 cm against DBH = 40.6 cm in Aldeia dos Fernandes, and were highly sensitive in their stem diameter decrement along two consecutives full growing seasons. This is the first exploratory study on assessing the stem diameter fluctuations on holm oaks to address a decline pathway. Our results revealed that holm oaks can survive during two consecutive growing seasons, with a continuous decrease in their stem diameters, which might indicate one tree’s decline pathway.

Cork rings have been extensively used in dendroecological studies of the Mediterranean evergreen cork oak ( Quercus suber L.). Through measurements of cork-ring width only, strong relationships have been found between cork-ring widths and climate parameters. To our knowledge, cork-ring density, which is an important cork quality attribute, has never been used in any dendroecological study to explore physiological responses of the cork oak to climate change. In this study, we measured cork-ring width and density over 50 years (1962–2013), corresponding to five consecutive cork harvests, and analyzed their inter-annual fluctuations in eight trees from two different sites, a wetter peneplain area (Benavente) and a drier mountainous area (Grândola). Our results revealed a statistically significant correlation between cork-ring width and density ( p < 0.05) at both sites; however, it was negative (r =− 0.261) and relatively weaker at Benavente, and positive at Grândola ( r = 0.410). Moreover, a direct relationship between cork density and spring–summer temperature was found only at Grândola. Here, cork growth sensitivity to inter-annual variability of precipitation and temperature in summer-autumn of the current growth year ( p < 0.01) was the highest and, respectively, positive and negative. We conclude that in drier Mediterranean environments, summer drought stress might negatively affect both cork growth and density, by changing tree physiological processes involved in cork formation, resulting in narrower and denser cork rings. Considering scenarios of future climate change effects in Southern Europe, in particular the longer drought periods and rising temperatures, it seems that both cork yield and cork density will be affected, with implications on cork quality.

As climate change increasingly affects forest ecosystems, detailed understanding of major effects is important to anticipate their consequences under future climate scenarios.The Mediterranean region is a prominent climate change hotspot, and evergreen cork oak (Quercus suber L.) woodlands are particularly climatically sensitive due to cork (bark) harvesting.Cork oak's drought avoidance strategy is well-known and includes structural and physiological adaptations that maximise soil water uptake and transport and limit water use, potentially leading to reduced stem and cork growth.Trees' responses to cope with water-limited conditions have been extensively described based on cork-rings width and, more recently, on cork-rings density, in dendroecological studies.However, so far, tree functional attributes and physiological strategies, namely photosynthetic metabolism adjustments affecting cork formation, have never been addressed and/or integrated on these previous cork-rings-based studies.In this study, we address the relation between carbon and oxygen stable isotopes of cork rings and precipitation and temperature, in two distinct locations of southwestern Portugal -the (wetter) Tagus basin peneplain and the (drier) Grândola mountains.We aimed at assessing whether the two climatic factors affect cork-ring isotopic composition under contrasting conditions of water availability, and, therefore, if carbon and oxygen signatures in cork can reflect tree functional (physiological and structural) responses to stressful conditions, which might be aggravated by climate change.Our results indicate differences between the study areas.At the drier site, the stronger statistically significant negative cork δ 13 C correlations were found with mean

Cork-ring widths have been extensively used in dendroecological studies assessing the relationship between cork growth patterns and climate (precipitation and temperature). Generally, cork growth is assumed as a proxy for stem diameter growth to address cork oak (Quercus suber L.) growth sensitivity to climate and cork yield modeling. Cork growth represents a large part of stem radial increment in this species due to the enhanced activity of phellogen when compared to the cambium activity; thus, similar inter-annual variations of cork-ring widths and tree diameter growth might be expected. However, so far, the influence of rainfall and temperature on stem diameter growth has scarcely been addressed; moreover, it is still not clear whether tree size relates, and in what way, to the variations in radial growth of cork and stem diameter and whether these reflect (proportional) quantitative variations in stem basal area growth. In this study, we computed the annual growth of cork and of stem diameter at breast-height in data series of 47 trees, from 2000 to 2012, corresponding to a full cork production cycle. Results showed a tight link between cork-ring width and stem diameter growth indices. However, while cork growth strongly correlated with climate conditions in autumn–winter prior to the growing season, stem diameter growth correlated with climate conditions of the current growing season, and, more importantly, it was tree size-related. The extrapolation from cork-ring increments to stem basal area growth is likely to progressively underestimate tree growth and biomass increment in larger cork oaks and to further bias it due to climate change effects in the Mediterranean region.

Climate change is impacting locally adapted species such as the keystone tree species cork oak ( Quercus suber L.). Quantifying the importance of environmental variables in explaining the species distribution can help build resilient populations in restoration projects and design forest management strategies. Using landscape genomics, we investigated the population structure and ecological adaptation of this tree species across the Mediterranean Basin. We applied genotyping by sequencing and derived 2,583 single nucleotide polymorphism markers genotyped from 81 individuals across 17 sites in the studied region. We implemented an approach based on the nearest neighbour haplotype ‘coancestry’ and uncovered a weak population structure along an east–west climatic gradient across the Mediterranean region. We identified genomic regions potentially involved in local adaptation and predicted differences in the genetic composition across the landscape under current and future climates. Variants associated with temperature and precipitation variables were detected, and we applied a nonlinear multivariate association method, gradient forest, to project these gene–environment relationships across space. The model allowed the identification of geographic areas within the western Mediterranean region most sensitive to climate change: south‐western Iberia and northern Morocco. Our findings provide a preliminary assessment towards a potential management strategy for the conservation of cork oak in the Mediterranean Basin.

This work focuses on the importance of considering the bio-substances evaluation of controversial strains against plant pathogens. The bacterial population of plant rhizosphere covers every type of plant-microbe interaction. Some of them have beneficial effects on plant growth and showed antagonistic activities against plant pathogens. The distribution of the Burkholderia genus is wide in soils, and many bacterial strains were proven to benefit and protect important crops from pathogen threats. In this study, it was possible to obtain a Burkholderia cenocepacia isolate from the rhizosphere soil of a leguminous plant. It was screened in vitro for its abilities to protect Trifolium subterraneum L. from Phytophthora cinnamomi. The current results revealed that plants enhanced the growth in plants inoculated with both, the bacterial strain and the pathogen probably because the bacteria produced antagonistic compounds such as hydrolytic enzymes. This report shows the potential ability of a rhizobacterium naturally existing in the soils to suppress the effects of the diseases caused by P. cinnamomi.

The thickness of bark (cork) is of utmost importance for its industrial processing and, therefore, for the sustainable management of cork oak (Quercus suberL.) woodlands. Cork thickness has been reported to decrease from the stem base upwards but, to our knowledge, prediction models have not yet been presented. This study intended to develop reliable models to predict stem profiles of cork thickness. We addressed the relationship between bark thickness and stem height by measuring stem diameters and cork thickness at various stem heights in 76 trees. Through a repeated measure ANOVA, we selected the bark thickness response variables, cork-ring width and cork thickness. A mixed-effect modelling approach was used to fit linear bark thickness curves with stem height. Our results showed that the equations that performed better for cork thickness were functions of the stem relative height and cross-sectional cork area increment. The decreasing trend upwards the stem might lead to underestimation of cork thickness at the base, more than overestimate it at upper stem parts, in relation to the values at breast height, which are generally used to estimate the thickness of the whole cork plank. Furthermore, cork thickness curves were tree size-related, with larger trees exhibiting a steeper reduction in cork thickness upwards the stem.

Thickness and porosity are properties of raw (natural) cork planks that have strong influence on the end-product quality, particularly on natural cork stoppers or discs, both highly valued natural cork products. In cork oak (Quercus suber L.), the between-trees variability of cork quality (thickness and porosity) is relatively well known and has been extensively studied. However, the within-tree variability of these properties has been scarcely addressed in the literature. In this study, we have focused on the within-tree variability of cork quality. Our major working hypothesis is that the within-tree variability of cork quality is related with trees specific growth patterns. We assessed, at tree individual level, the stem height-cork thickness and the stem height-cork porosity relationships that, so far, were not clear. Eventually, the patterns of radial growth, along the stem upwards may have a strong physiological basis. Our results showed: i) a stem height-cork thickness relationship, with trees showing a decrease of the cork thickness with the stem height and; (ii) a stem height-cork porosity relationship, with trees showing a decrease in the cork porosity with the stem height. Forest managers and cork processors require more quantitative information on the inter- and intra-stem variation of cork-thickness and cork-porosity in order to understand the effects of forest management decisions at cork oak woodlands level. This way, the modeled stem profiles of cork quality (thickness and porosity) allow the optimization of high quality raw cork (planks) and determine the economic feasibility of cork harvesting practices.

Cork cambium (or phellogen) is a secondary meristem responsible for the formation of phelloderm and phellem/cork, which together compose the periderm. In Quercus suber L., the phellogen is active throughout the entire life of the tree, producing a continuous and renewable outer bark of cork. To identify specific candidate genes associated with cork cambium activity and phellem differentiation, we performed a comparative transcriptomic study of Q. suber secondary growth tissues (xylem and phellogen/phellem) using RNA-seq. The present work provides a high-resolution map of all the transcripts identified in the phellogen/phellem tissues. A total of 6013 differentially expressed genes were identified, with 2875 of the transcripts being specifically enriched during the cork formation process versus secondary xylem formation. Furthermore, cork samples originating from the original phellogen ('virgin' cork) and from a traumatic phellogen ('amadia' cork) were also compared. Our results point to a shortlist of potentially relevant candidate genes regulating phellogen activity and phellem differentiation, including novel genes involved in the suberization process, as well as genes associated to ethylene and jasmonate signaling and to meristem function. The future functional characterization of some of the identified candidate genes will help to elucidate the molecular mechanisms underlying cork cambium activity and phellem differentiation.

The cork oak (Quercus suber L.) is periodically harvested for bark (cork) throughout its lifetime. Trees undergo physiological changes as they age which affect stem diameter growth and their sensitivity to climate. However, little is known about trees age- or size-related growth changes and it remains unclear if trees of different ages (sizes) have similar climate-growth relationships. In this study, we examined the increment in stem basal area of 47 randomly selected (large and small) cork oaks over a 12-year period to assess divergent climate-growth relationships. Our approach, using a machine learning algorithm on unlabelled data sets of basal area increments, successfully filtered out tree-clusters that suggested a size (age)-dependent growth response to climate. On average, the basal area increment was more than three times larger in larger-trees clusters compared with smaller-trees clusters. A large tree (diameter >75 cm) on average added 105 cm(2) y(-1) to its basal area against 25 cm(2) y(-1) in a small tree (diameter <35 cm). Additionally, in smaller-trees, cork harvesting intensified the negative impact of drought on tree growth, and worsened post-drought recovery. These findings highlight the need to consider biological growth trends for accurate predictions of trees responses to drought.

Europe’s woodland and savanna rangelands, often part of silvopastoral systems known as wood-pastures, are deteriorating because of abandonment that leads to return to a forested state or lack of tree regeneration from overgrazing or tree and shrub removal. Despite numerous local studies, there has been no broader survey of the stand structure of European wood-pastures showing which systems are at risk of losing their semiopen character. This overview aims to 1) show some of the differences and similarities in wood-pastures from landscapes across Europe and 2) identify which of these wood-pastures are at risk of losing their semiopen character. We collated a dataset of 13 693 trees from 390 plots in wood-pastures from eight different European regions (western Estonia, eastern Greece, northern Germany, Hungary, northern Italy, southern Portugal, central Romania, and southern Sweden), including tree diameters at breast height, tree density, management type, and tree species composition. On the basis of their structural characteristics, we classified wood-pastures using principal component analysis (PCA) and cluster analysis. The PCA showed a gradient from dense wood-pastures with high levels of regeneration (e.g., in Estonia) to sparse wood-pastures with large trees but a lack of regeneration (e.g., in Romania). Along this gradient, we identified three main groups of wood-pastures: 1) sparse wood-pastures with mostly big trees; 2) dense wood-pastures composed of small trees, and 3) wood-pastures containing a wide range of tree ages. Our results show a large structural gradient in European wood-pastures, as well as regeneration problems varying in their severity, highlighting the importance of social-ecological context for wood-pasture conditions. To maintain the ecological and cultural integrity of European wood-pastures, we suggest 1) more comprehensively considering them in European policies such as the Common Agricultural Policy and EU Habitats Directive, while 2) taking into account their structural characteristics and social-ecological backgrounds.

In temperate regions, the analysis of climate signals encoded in the wood rings allows assessing tree sensitivity to climate and its potential effects on growth and yield. In Mediterranean regions, the cork oak (Quercus suber L.) has received limited attention for dendrochronological studies because tree rings are faint and cork rings with a clear annual banding have been rather neglected. We analyzed the climatic signal of cork-ring chronologies for AD 1996-2010 from distinct regions in western Algeria [a mountain oak forest called Hafir-Zarieffet] and in southern Portugal [a peneplain wood-pasture called Benavente]. The goal was to evaluate the strength and consistency of climate signal and to assess cork growth sensitivity to climate variables (precipitation and temperature). Our results suggest that cork growth encodes a climatic signal. Trends of cork growth, via correlation analysis, including climate variables (on a monthly and seasonal basis) and Lang's index (on an annual basis) were successfully assessed. Drought-driven cork growth reduction is a threshold function of a P-T ratio, and with expected increase in the drought occurrence under changing climate, cork growth is likely to be similarly affected in both regions.

DNA methylation is thought to influence Quercus suber cork quality, which is the main constraint for its economic valorisation. However, a deep knowledge of the cytosine methylation patterns disclosing the epigenetic variability of trees with different cork quality types is totally missing. This study investigates the hypothesis that variations in DNA methylation contribute to differences in cork cellular characteristics directly related to original or traumatic phellogen activity. We used MSAPs (Methylation Sensitive Amplified Polymorphism) to assess DNA methylation patterns of cork and leaf tissues of Q. suber adult trees growing in three cork oak stands. The relationship between the detected polymorphisms and the diversity of cork quality traits was explored by a marker-trait analysis focusing on the most relevant quality characteristics. Populations differed widely in cork quality, but only slightly in degree of epigenetic differentiation. Four MSAP markers (1.3% of the total) were significantly associated with the most noteworthy quality traits: wood inclusions (nails) and porosity. This evidence supports the potential role of cytosine methylation in the modulation of differential phellogen activity either involved in localized cell death or in pore production, resulting in different cork qualities. Although, the underlying basis of the methylation polymorphism of loci affecting cork quality traits remain unclear, the disclosure of markers statistically associated with cork quality strengthens the potential role of DNA methylation in the regulation of these traits, namely at the phellogen level.

The sol-gel method was employed in the preparation of organic/inorganic biohybrid ormolytes composed of a di-urethane cross-linked poly(epsilon-caprolactone) (PCL(530))/siloxane matrix doped for the first time with different amounts of sodium triflate (NaCF3SO3.xH(2)O). The samples are semi-crystalline and thermally stable up to ca. 250 degrees C. The maximum ionic conductivity (4.1 x 10(-7), 1.5 x 10(-4) and 8.8 x 10(-4) S cm(-1) at 30, 49 and 97 degrees C, respectively) was attained at n = 6, where n (composition) corresponds to the number of ester (C(double bond; length as m-dashO)(CH2)5O) repeat units of PCL(530) per sodium ion. Selected ormolytes were employed in the construction and characterization of prototype electrochromic devices (ECDs). Tests with ECDs incorporating poly(oxyethylene)/siloxane (U(600))(n)NaCF3SO3 (where n is the ratio of ether (OCH2CH2) repeat units per sodium ion) ormolytes already reported were also performed. The devices exhibited fast switching time of ca. 30 s. At 555 nm the ECD@U600(63)Na device exhibited the highest optical density value (Delta(OD) = 0.12) and coloration efficiency for bleaching (CE(ou)t 131 cm(2)C-1, 50th cycle) and good optical memory. The ECD@PCL58Na device exhibited a CE value for coloration (0.12 versus 0.061) similar to that of ECD@U600(63)Na (CEin -116 cm2C-1, 50th cycle) and a very good stability. However the Delta(OD) value for the latter device was much higher.

In the Mediterranean climate regions, drought events are expected to affect the growth of forests ecosystems by changing trees growth rates and eventually inducing shifts in their growth patterns. Cork oak (Quercus suber L.) is a strictly western Mediterranean tree species periodically harvested for its bark, the cork. So far, cork oak has received limited attention for dendroclimatological studies due to its typical faint and erratic tree wood rings. Moreover, its distinct cork rings chronologies have been completely neglected. In this study we introduce an approach using cork ring chronologies dated back 9–10 years for climate response. Despite enhancing interannual variability and increasing statistical response to short-term climatic variability, still poorly understood, this study will possibly allow infer long-term climate response. We analyzed the cork ring chronologies of 55 cork samples collected in mature (under exploitation) trees in three distinct locations in southwestern Portugal. Cork growth recorded a high climate signal, with highly significant and coherent responses to the yearly climate-related sources of variation. We successfully assessed trends of cork growth via correlation analysis including selected climate variables among mean monthly temperature, monthly precipitation and, on an annual basis, eight precipitation indices. The high mean sensitivities and inter-series correlations found for cork ring chronologies combined with the significant variance explained by climate variables suggest that climate is likely one dominant signal that affects cork growth, but local environmental stresses can decisively affect this (climate) signal. Assuming cork growth as a proxy for cork oak growth, it seems conceivable that despite the trees being highly resistant to drought stress, cork oak woodlands in southwestern Portugal would have to face lesser growth in a global warming scenario.

In the southwestern Mediterranean Basin, cork oaks (Quercus suber L.) are periodically harvested for their cork. This natural product is valued by its homogeneity which heightens the importance of characterizing cork tissue discontinuities, or cork pores. Cork porosity profile in natural cork planks has been reported to be affected by forest management practices but, so far, has been scarcely addressed. We characterize the cork porosity profile in two contrasting cork oak woodland; at a mountain forest, in Western Algeria (absence of forest management) and at a peneplain "montado," in southern Portugal (intensively managed toward the optimization of cork production). Image analysis techniques were applied on transverse sections of more than 40 cork samples from both woodland, and a stepwise discriminant analysis was used to discriminate between the cork pore features datasets. Cork porosity profiles were similar between regions but; in the cork samples from Algeria, cork pores were having higher values for linear dimensions of pores (length and perimeter) and contrasting shape values (roundness) which depreciate cork quality, when compared to the cork samples from Portugal. However, improved woodland management strategies at Algeria should ensure adequate cork homogeneity and suitability for more valuable cork products.

Cork oak (Quercus suber L.) woodlands in the Mediterranean basin may cope with drought continuing to grow during the dry summer period. This growth pattern relies on the presence of extensive and deep root systems and on their access to groundwater. When the water table level is not within the reach of roots or decreases below a certain level following periods of drought, tree growth and survival might be affected. So far, little is known about the sensitivity and resilience of cork oak to both the rising and lowering of the water table over a long-term period, e.g., during one cork production cycle (9-10 years). In this study, we determined whether changes in groundwater levels influence cork growth in two cork oak woodlands located at the Tagus alluvium aquifer in southwestern Portugal. The selected areas have contrasting groundwater levels and an integrated framework was developed combining: (a) a standardized annual cork growth index series for tree (cork) growth assessment; (b) a monthly Standardized Precipitation Evapotranspiration Index (SPEI) for drought periods assessment and; (c) a Geographical Information System (GIS)-based model for mapping the mean water table depth in the dry period. Results showed a decrease of cork growth index (up to 40%) immediately following a severe drought, which lowered the water table in the dry period. Moreover, in the site with deeper groundwater levels the reduction of cork growth index was higher. The mean water table depth (in dry period) of -2.5 m was found to be a trigger value for cork growth. Our findings suggest that, under shallow water table conditions groundwater mitigates the seasonal fluctuations of precipitation and trees may use groundwater to decouple cork growth from the annual precipitation regime. We conclude that, under changing Mediterranean environments, with expected increase in drought events, the decrease of groundwater levels may severely affect cork (and tree) growth and ultimately tree survival. The management and conservation of cork oak woodlands should therefore rely on adequate policies for the sustainability of groundwater resources. (C) 2016 Elsevier B.V. All rights reserved.