Restoring oak forests through direct seeding or planting: Protocol for a continental-scale experiment.

The choice of revegetating via direct seeding or planting nursery-grown seedlings influences the potential stresses suffered by seedlings such as herbivory and drought. The outcome of the balance between both revegetation methods may ultimately depend on how species identity and traits such as seed and seedling size interact with environmental conditions. To test this, we will conduct a continental-scale experiment consisting of one mini-experiment replicated by multiple participants across Europe. Each participant will establish a site with seeded and planted individuals of one or more native, locally growing oak (Quercus) species; the selection of this genus aims to favour continental-scale participation and to allow testing the response of a widely distributed genus of broad ecological and economic relevance. At each site, participants will follow the present protocol for seed collection, seeding in the field, nursery cultivation, outplanting, protection against herbivores, site maintenance, and measurement of seedling performance and environmental variables. Each measurement on each species at each site will produce one effect size; the data will be analysed through mixed-effects meta-analysis. With this approach we will assess the main effect of revegetation method, species, plant functional traits, and the potential effect of site-specific effect moderators. Overall, we will provide a continental-scale estimate on the seeding vs. planting dilemma and analyse to what extent the differences in environmental conditions across sites, seed size, functional traits, and the phylogenetic relatedness of species can account for the differences in the effect of revegetation method on seedling performance across study sites and species.

Climate and species stress resistance modulate the higher survival of large seedlings in forest restorations worldwide.

Seedling planting plays a key role in active forest restoration and regeneration of managed stands. Plant attributes at outplanting can determine tree seedling survival and consequently early success of forest plantations. Although many studies show that large seedlings of the same age within a species have higher survival than small ones, others report the opposite. This may be due to differences in environmental conditions at the planting site and in the inherent functional characteristics of species. Here, we conducted a global-scale meta-analysis to evaluate the effect of seedling size on early outplanting survival. Our meta-analysis covered 86 tree species and 142 planting locations distributed worldwide. We also assessed whether planting site aridity and key plant functional traits related to abiotic and biotic stress resistance and growth capacity, namely specific leaf area and wood density, modulate this effect. Planting large seedlings within a species consistently increases survival in forest plantations worldwide. Species' functional traits modulate the magnitude of the positive seedling size-outplanting survival relationship, showing contrasting effects due to aridity and between angiosperms and gymnosperms. For angiosperms planted in arid/semiarid sites and gymnosperms in subhumid/humid sites the magnitude of the positive effect of seedling size on survival was maximized in species with low specific leaf area and high wood density, characteristics linked to high stress resistance and slow growth. By contrast, high specific leaf area and low wood density maximized the positive effect of seedling size on survival for angiosperms planted in subhumid/humid sites. Results have key implications for implementing forest plantations globally, especially for adjusting nursery cultivation to species' functional characteristics and planting site aridity. Nursery cultivation should promote large seedlings, especially for stress sensitive angiosperms planted in humid sites and for stress-resistant species planted in dry sites.

Normalization criteria determine the interpretation of nitrogen effects on the root hydraulics of pine seedlings.

Plant hydraulics is key for plant survival and growth because it is linked to gas exchange and drought resistance. Although the environment influences plant hydraulics, there is no clear consensus on the effect of nitrogen (N) supply, which may be, in part, due to different hydraulic conductance normalization criteria and studied species. The objective of this study was to compare the variation of root hydraulic properties using several normalization criteria in four pine species in response to three contrasting N fertilization regimes. We studied four closely related, yet ecologically distinct species: Pinus nigra J.F. Arnold, Pinus pinaster Ait., Pinus pinea L. and Pinus halepensis Mill. Root hydraulic conductance (Kh) was measured with a high-pressure flow meter, and values were normalized by total leaf area (leaf specific conductance, Kl), xylem cross-section area (xylem specific conductance, Ks), total root area (root specific conductance, Kr) and the area of fine roots (fine root specific conductance, Kfr). Controlling for organ size differences allowed comparison of the hydraulic efficiency of roots to supply or absorb water among fertilization treatments and species. The effect of N on the root hydraulic efficiency depended on the normalization criteria. Increasing N availability reduced Kl and Ks, but increased Kh, Kr and especially Kfr. The positive effect of N on Kr and Kfr was positively related to seedling relative growth rate and was also consistent with published results at the interspecific level, whereby plant hydraulics is positively linked to photosynthesis and transpiration rate and fast growth. In contrast, normalization by leaf area and xylem cross-sectional area (Kl and Ks) reflected opposite responses to Kr and Kfr. This indicates that the normalization criteria determine the interpretation of the effect of N on plant hydraulics, which can limit species and treatment comparisons.

Moderate water stress does not inhibit nitrogen remobilization, allowing fast growth in high nitrogen content Quercus variabilis seedlings under dry conditions.

Remobilization of stored nitrogen (N) plays an important role in the early growth of deciduous trees in spring. Several environmental factors can modulate N remobilization, but whether water stress is one such factors is unknown. This study analyzes how the size of N storage in Quercus variabilis Blume seedlings interacts with water stress to affect N remobilization, uptake and new growth. This information is important for improving success of forest tree plantations under dry spring conditions. During the first growing season, we produced seedlings with distinct N content by applying two fall N fertilization rates (12 or 24 mg N per seedling) using 15N-enriched fertilizer. At the beginning of the second growing season, a new experiment was started where seedlings were transplanted into larger pots and subjected to two watering levels (85 or 40% of field capacity). The plants were sampled at 4 weeks (T1), 8 weeks (T2) and 12 weeks (T3) after transplanting. Low watering reduced the growth of high and low N seedlings, but high N seedlings showed greater growth than low N seedlings. During bud burst and initial shoot elongation (T1), restricted watering, which induced a moderate water stress, did not affect the amount of N remobilized from roots, the major source of stored N source at this growth stage. This suggests that high N storage can partially counteract the negative effect of moderate water stress on early growth. At T1, water stress did not affect N uptake, and high N content seedlings absorbed significantly less soil N than did low N content seedlings. At T3, in contrast, water stress was the main determinant for N uptake, with drought-stressed plants showing lower uptake than well-watered plants. We conclude that moderate drought does not inhibit N remobilization from the major storage organ at early growth stages in spring, and that increasing N storage of planted seedlings through fall fertilization can mitigate the negative effect of moderate spring drought on growth.

The "isohydric trap": A proposed feedback between water shortage, stomatal regulation, and nutrient acquisition drives differential growth and survival of European pines under climatic dryness.

Climatic dryness imposes limitations on vascular plant growth by reducing stomatal conductance, thereby decreasing CO2 uptake and transpiration. Given that transpiration-driven water flow is required for nutrient uptake, climatic stress-induced nutrient deficit could be a key mechanism for decreased plant performance under prolonged drought. We propose the existence of an "isohydric trap," a dryness-induced detrimental feedback leading to nutrient deficit and stoichiometry imbalance in strict isohydric species. We tested this framework in a common garden experiment with 840 individuals of four ecologically contrasting European pines (Pinus halepensis, P. nigra, P. sylvestris, and P. uncinata) at a site with high temperature and low soil water availability. We measured growth, survival, photochemical efficiency, stem water potentials, leaf isotopic composition (δ13 C, δ18 O), and nutrient concentrations (C, N, P, K, Zn, Cu). After 2 years, the Mediterranean species Pinus halepensis showed lower δ18 O and higher δ13 C values than the other species, indicating higher time-integrated transpiration and water-use efficiency (WUE), along with lower predawn and midday water potentials, higher photochemical efficiency, higher leaf P, and K concentrations, more balanced N:P and N:K ratios, and much greater dry-biomass (up to 63-fold) and survival (100%). Conversely, the more mesic mountain pine species showed higher leaf δ18 O and lower δ13 C, indicating lower transpiration and WUE, higher water potentials, severe P and K deficiencies and N:P and N:K imbalances, and poorer photochemical efficiency, growth, and survival. These results support our hypothesis that vascular plant species with tight stomatal regulation of transpiration can become trapped in a feedback cycle of nutrient deficit and imbalance that exacerbates the detrimental impacts of climatic dryness on performance. This overlooked feedback mechanism may hamper the ability of isohydric species to respond to ongoing global change, by aggravating the interactive impacts of stoichiometric imbalance and water stress caused by anthropogenic N deposition and hotter droughts, respectively.

Distribution of pines in the Iberian Peninsula agrees with species differences in foliage frost tolerance, not with vulnerability to freezing-induced xylem embolism.

Drought and frosts are major determinants of plant functioning and distribution. Both stresses can cause xylem embolism and foliage damage. The objective of this study was to analyse if the distribution of six common pine species along latitudinal and altitudinal gradients in Europe is related to their interspecific differences in frost tolerance and to the physiological mechanisms underlying species-specific frost tolerance. We also evaluate if frost tolerance depends on plant water status. We studied survival to a range of freezing temperatures in 2-year-old plants and assessed the percentage loss of hydraulic conductivity (PLC) due xylem embolism formation and foliage damage determined by needle electrolyte leakage (EL) after a single frost cycle to -15 °C and over a range of predawn water potential (ψpd) values. Species experiencing cold winters in their range (Pinus nigra J.F. Arnold, Pinus sylvestris L. and Pinus uncinata Raymond ex A. DC.) had the highest frost survival rates and lowest needle EL and soluble sugar (SS) concentration. In contrast, the pines inhabiting mild or cool winter locations (especially Pinus halepensis Mill. and Pinus pinea L. and, to a lesser extent, Pinus pinaster Ait.) had the lowest frost survival and highest needle EL and SS values. Freezing-induced PLC was very low and differences among species were not related to frost damage. Reduction in ψpd decreased leaf frost damage in P. pinea and P. sylvestris, increased it in P. uncinata and had a neutral effect on the rest of the species. This study demonstrates that freezing temperatures are a major environmental driver for pine distribution and suggests that interspecific differences in leaf frost sensitivity rather than vulnerability to freezing-induced embolism or SS explain pine juvenile frost survival.

Species ecology determines the role of nitrogen nutrition in the frost tolerance of pine seedlings.

Frost determines the evolution and distribution of plants in temperate and cold regions. Several environmental factors can influence frost acclimation of woody plants but the magnitude and direction of the effect of nitrogen (N) availability is controversial. We studied the effect of N availability on root and shoot frost tolerance in mid-fall and in winter in seedlings of four pines of contrasting ecology: Pinus nigra J.F. Arnold, P. pinaster Ait., P. pinea L. and P. halepensis Mill.. Organ N and soluble sugar concentration, and timing of cessation of shoot elongation were measured to assess the physiological mechanisms underlying frost acclimation. Nitrogen was supplied at high and low rates only during the pre-hardening period and at a moderate N rate during hardening in the fall. Shoot frost tolerance increased over winter while root frost tolerance did not change in any species. Pre-hardening N availability affected the frost tolerance of both roots and shoots, although the effect was species-specific: high N reduced the overall root and shoot frost tolerance in P. pinea and P. halepensis, and increased the frost tolerance in P. nigra, but had no effect in P. pinaster. Nitrogen supply in the fall consistently increased frost tolerance in all species. Differences in frost tolerance among species and N treatments were not explained by variations in organ N or soluble carbohydrate concentration, nor by timing of cessation of shoot elongation; however, the most frost tolerant species ceased elongation earlier than the least frost tolerant species. Despite the close phylogenetic relatedness of the studied species, the effect of N availability on seedling frost tolerance differed among species, indicating that species ecology (especially frost acclimation physiology) and timing of N supply drives the effect of N availability on frost tolerance of pine species.

Effective nut dispersal by magpies (Pica pica L.) in a Mediterranean agroecosystem.

Scatter-hoarding animals such as corvids play a crucial role in the dispersal of nut-producing tree species. This interaction is well known for some corvids, but remains elusive for other species such as the magpie (Pica pica), an abundant corvid in agroecosystems and open landscapes of the Palearctic region. In addition, the establishment of the individual dispersed seeds-a prerequisite for determining seed-dispersal effectiveness-has never before been documented for the interaction between corvids and nut-producing trees. We analyzed walnut dispersal by magpies in an agroecosystem in southern Spain. We used several complementary approaches, including video recording nut removal from feeders, measuring dispersal distance using radio tracking (with radio transmitters placed inside nuts), and monitoring the fate of dispersed nuts to the time of seedling emergence. Magpies were shown to be highly active nut dispersers. The dispersal distance averaged 39.6 ± 4.5 m and ranged from 4.1 to 158.5 m. Some 90% of the removed walnuts were cached later, and most of these (98%) were buried in the soil or hidden under plant material. By the time of seedling emergence, ca. 33% of nuts remained at the caching location. Finally, 12% of the cached nuts germinated and 4% yielded an emerged seedling, facilitating the transition to the next regeneration stage. The results demonstrate for the first time that magpies can be an effective scatter-hoarding disperser of a nut-producing tree species, suggesting that this bird species may play a key role in the regeneration and expansion of broadleaf forests in Eurasia.

Fast growth involves high dependence on stored resources in seedlings of Mediterranean evergreen trees.

BACKGROUND AND AIMS: The carbon (C) and nitrogen (N) needed for plant growth can come either from soil N and current photosynthesis or through remobilization of stored resources. The contribution of remobilization to new organ growth on a whole-plant basis is quite well known in deciduous woody plants and evergreen conifers, but this information is very limited in broadleaf evergreen trees. This study compares the contribution of remobilized C and N to the construction of new organs in spring, and assesses the importance of different organs as C and N sources in 1-year-old potted seedlings of four ecologically distinct evergreen Mediterranean trees, namely Quercus ilex, Q. coccifera, Olea europaea and Pinus hapelensis. METHODS: Dual (13)C and (15)N isotope labelling was used to unravel the contribution of currently taken up and stored C and N to new growth. Stored C was labelled under simulated winter conditions. Soil N was labelled with the fertilization during the spring growth. KEY RESULTS: Oaks allocated most C assimilated under simulated winter conditions to coarse roots, while O. europaea and P. halepensis allocated it to the leaves. Remobilization was the main N source (>74 %) for new fine-root growth in early spring, but by mid-spring soil supplied most of the N required for new growth (>64 %). Current photosynthesis supplied >60 % of the C in new fine roots by mid-spring in most species. Across species, the proportion of remobilized C and N in new shoots increased with the relative growth rate. Quercus species, the slowest growing trees, primarily used currently acquired resources, while P. halepensis, the fastest growing species, mainly used reserves. Increases in the amount of stored N increased N remobilization, which fostered absolute growth both within and across species. Old leaves were major sources of remobilized C and N, but stems and roots also supplied considerable amounts of both in all species except in P. halepensis, which mainly relied on foliage formed in the previous growing season to supply stored resources. CONCLUSIONS: Seedlings of Mediterranean evergreen trees have distinct C and N storage physiologies, with relative growth rate driving the contribution of remobilized resources to new growth. These differences may reduce competition and facilitate species coexistence.

Nitrogen nutrition and drought hardening exert opposite effects on the stress tolerance of Pinus pinea L. seedlings.

Functional attributes determine the survival and growth of planted seedlings in reforestation projects. Nitrogen (N) and water are important resources in the cultivation of forest species, which have a strong effect on plant functional traits. We analyzed the influence of N nutrition on drought acclimation of Pinus pinea L. seedlings. Specifically, we addressed if high N fertilization reduces drought and frost tolerance of seedlings and whether drought hardening reverses the effect of high N fertilization on stress tolerance. Seedlings were grown under two N fertilization regimes (6 and 100 mg N per plant) and subjected to three drought-hardening levels (well-watered, moderate and strong hardening). Water relations, gas exchange, frost damage, N concentration and growth at the end of the drought-hardening period, and survival and growth of seedlings under controlled xeric and mesic outplanting conditions were measured. Relative to low-N plants, high-N plants were larger, had higher stomatal conductance (27%), residual transpiration (11%) and new root growth capacity and closed stomata at higher water potential. However, high N fertilization also increased frost damage (24%) and decreased plasmalemma stability to dehydration (9%). Drought hardening reversed to a great extent the reduction in stress tolerance caused by high N fertilization as it decreased frost damage, stomatal conductance and residual transpiration by 21, 31 and 24%, respectively, and increased plasmalemma stability to dehydration (8%). Drought hardening increased tissue non-structural carbohydrates and N concentration, especially in high-fertilized plants. Frost damage was positively related to the stability of plasmalemma to dehydration (r = 0.92) and both traits were negatively related to the concentration of reducing soluble sugars. No differences existed between moderate and strong drought-hardening treatments. Neither N nutrition nor drought hardening had any clear effect on seedling performance under xeric outplanting conditions. However, fertilization increased growth under mesic conditions, whereas drought hardening decreased growth. We conclude that drought hardening and N fertilization applied under typical container nursery operational conditions exert opposite effects on the physiological stress tolerance of P. pinea seedlings. While drought hardening increases overall stress tolerance, N nutrition reduces it and yet has no effect on the drought acclimation capacity of seedlings.

Remobilization of acorn nitrogen for seedling growth in holm oak (Quercus ilex), cultivated with contrasting nutrient availability.

The relative contribution of nitrogen (N) reserves from seeds or uptake by the roots to the growth and N content of young seedlings has received little attention. In this study, we investigated the contribution of N from the acorn or uptake by the roots to the N content of holm oak (Quercus ilex L.) seedlings and determined if remobilization of acorn N was affected by nutrient availability in the growing media. Q. ilex seedlings were cultivated for 3 months, until the end of the second shoot flush of growth, with three N fertilization rates: 8.6 mM N, 1.4 mM N or no fertilization. Fertilizer N was enriched in (15)N. Between 62 and 75% of the N contained in high and low fertilized seedlings, respectively, at the end of the second flush of growth was derived from the acorn. However, the dependence on acorn N was greater during the early root growth and first shoot flush of growth and decreased during the second shoot flush of growth, with root uptake contributing 32-54% of plant new N in this latter developmental stage in high and low fertilized plants, respectively. Fertilization rate did not affect the amount of N taken up during the earliest developmental stages, but it increased it during the second shoot flush of growth. Fertilization increased the mass of the shoot segment formed during the second shoot flush of growth and reduced the root mass, with no effect on whole plant growth. Remobilization of acorn N was faster in unfertilized plants than in fertilized plants. It is concluded that the holm oak seedlings depend greatly upon acorn N until the end of the second shoot flush of growth, that significant root N uptake starts at the beginning of the second shoot flush of growth and that acorn N remobilization is a plastic process that is accelerated under extremely low substratum nutrient content.

Drought tolerance and transplanting performance of holm oak (Quercus ilex) seedlings after drought hardening in the nursery.

Drought stress is the main cause of mortality of holm oak (Quercus ilex L.) seedlings in forest plantations. We therefore assessed if drought hardening, applied in the nursery at the end of the growing season, enhanced the drought tolerance and transplanting performance of holm oak seedlings. Seedlings were subjected to three drought hardening intensities (low, moderate and severe) for 2.5 and 3.5 months, and compared with control seedlings. At the end of the hardening period, water relations, gas exchange and morphological attributes were determined, and survival and growth under mesic and xeric transplanting conditions were assessed. Drought hardening increased drought tolerance primarily by affecting physiological traits, with no effect on shoot/root ratio or specific leaf mass. Drought hardening reduced osmotic potential at saturation and at the turgor loss point, stomatal conductance, residual transpiration (RT) and new root growth capacity (RGC), but enhanced cell membrane stability. Among treated seedlings, the largest response occurred in seedlings subjected to moderate hardening. Severe hardening reduced shoot soluble sugar concentration and increased shoot starch concentration. Increasing the duration of hardening had no effect on water relations but reduced shoot mineral and starch concentrations. Variation in cell membrane stability, RT and RGC were negatively related to osmotic adjustment. Despite differences in drought tolerance, no differences in mortality and relative growth rate were observed between hardening treatments when the seedlings were transplanted under either mesic or xeric conditions.