A taxonomic tool for identifying needle remains of south‐western European Pinus species of the Late Quaternary

This work provides a tool whereby the needle remains of native, south‐western European Pinus spp. can be easily identified from species‐specific epidermal features. To construct this tool, the needles of P. uncinata, P. sylvestris, P. nigra, P. pinaster, P. pinea and P. halepensis were gathered across the Northern Hemisphere range of each taxon and compared with non‐indigenous trees growing in two South Australian Botanic Gardens. Three needles from each of these species were taken from three adult trees growing at three different localities. Light microscopy was used to observe the key epidermal and stomatal features of the needles. To improve interpretation, additional scanning electron microscopy samples were prepared. Epidermal features, including variation in the diameter of the epistomatal chamber aperture (pore), are described. A taxonomic key based on the size, shape and arrangement of the subsidiary cells of the stomatal complexes was constructed. This key enables the identification of pine needle fragments at the species level (except those belonging to the group P. gr. nigra‐uncinata). Despite their overlapping range, pore size was helpful in distinguishing between P. nigra and P. uncinata and between three groups of species. Isolated stomata were also observed. Cluster and discriminant analyses of stomatal variables described in earlier studies were performed. Overlap in guard cell variables hampers species‐level identification of isolated stomata. Species discrimination is improved if groups of ecological affinity are considered. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 282–298.     

Carbon isotope discrimination and stomatal responses of mature Pinus sylvestris L. trees exposed in situ for three years to elevated CO2 and temperature

The Climate Change Experiment (CLIMEX) is a unique large scale facility in which an entire undisturbed catchment of boreal vegetation has been exposed to elevated CO₂ (560 ppm) and temperature (+3°C summer, +5°C winter) for the past three years with all the soil-plant-atmosphere linkages intact. Here, carbon isotope composition and stomatal density have been analysed from sequential year classes of needles of mature Scots pine trees (Pinus sylvestris L.) to investigate the response of time-integrated water-use efficiency (UWE) and stomatal density to CO₂ enrichment and climate change. Carbon isotope discrimination decreased and WUE increased in cohorts of needles developing under increased CO₂ and temperature, compared to needles on the same trees developing in pretreatment years. Mid-season instantaneous gas exchange, measured on the same trees for the past four years, indicated that these responses resulted from higher needle photosynthetic rates and reduced stomatal conductance. Needles of P. sylvestris developing under increased CO₂ and temperature had consistently lower stomatal densities than their ambient grown counterparts on the same trees. The stomatal density of P. sylvestris needles was inversely correlated with δ¹³C-derived WUE, implying some effect of this morphological response on leaf gas exchange. Future atmospheric CO₂ and temperature increases are therefore likely to improve the water economy of P. sylvestris, at least at the scale of individual needles, by affecting stomatal density and gas exchange processes.     

Osservazioni sulla lunghezza e sulla longevità degli aghi di pino domestico (Pinus pinea L.)

Abstract

Observations on needle length and longevity of Pinus pinea L. - We investigated needle length and longevity of Pinus pinea L. to understand their ecological significance. Samplings regarded five coastal pinewoods (four in the province of Grosseto, one in the province of Lucca) and isolated trees in different sites of Tuscany. We found a significant variation in needle length among different positions of the crown (central, intermediate and peripheral), among individuals of the same site and among different sites; isolated trees showed longer needles and a longer retention than pines in stands. We also obtained a highly significant linear regression between length of different-aged needles (from 1988 to 1992) and rainfall of the period march-august of the same years. Our results suggest that, despite its high variability, needle ength is influenced by climatic conditions (overall by rainfalls) and by the water availability of the site where pines grow. Needle retention depends largely on the position of needles within the crown and on the drought effect: at the upper position, three or, seldom, four-year old needle can persist if the summer drought period is not too long and the site conditions are favourable; at the lower level only two-year old needles are retained. Needle longevity decreases sharply in years with severe drought, therefore it can be used as an index of climatic and edaphic stress.     

Differences between Pinus pinea and Pinus pinaster as bioindicators of polycyclic aromatic hydrocarbons

The potential of Univariate and Multivariate Analysis and specifically Principal Components Analysis (PCA), has been employed to assess the performance of pine needles as bioindicators of polycyclic aromatic hydrocarbons (PAHs) and particularly emphasize the crucial importance of making a distinction between different pine species if more than one is sampled. Four sampling sessions were done in 29 sites and needles of two common pine species (Pinus pinaster Ait. in 19 sites and Pinus pinea L. in 12) were collected and analysed using gas chromatography-mass spectrometry (GC-MS) and PCA. The results obtained indicated significant differences between species, attributed to their different morphology. The mean total PAH concentration of the P. pinaster needles are over two times higher than P. pinea's. This difference is lower when the results are presented in lipid weight, but still statistically significant. Samples from the two sites with adjacent trees reinforce these conclusions, showing significant differences in terms of PAH mean concentration and aromatic ring patterns.     

Why does photosynthesis decrease with needle age in Pinus pinaster?

Rates of photosynthesis vary with foliage age and typically decline from full-leaf expansion until senescence occurs. This age-related decline in photosynthesis is especially important in species that retain foliage for several years, yet it is not known whether the internal conductance to CO₂ movement (g _i) plays any role. More generally, g _i has been measured in only a few conifers and has never been measured in leaves or needles older than 1 year. The effect of ageing on g _i was investigated in Pinus pinaster, a species that retains needle for 4 or more years. Measurements were made in autumn when trees were not water limited and after leaf expansion was complete. Rates of net photosynthesis decreased with needle age, from 8 μmol m⁻² s⁻¹ in fully expanded current-year needles to 4.4 μmol m⁻² s⁻¹ in 3-year-old needles. The relative limitation due to internal conductance (0.24–0.35 out of 1) was in all cases larger than that due to stomatal conductance (0.13–0.19 out of 1). Internal conductance and stomatal conductance approximately scaled with rates of photosynthesis. Hence, there was no difference among year-classes in the relative limitations posed by internal and stomatal conductance or evidence that they cause the age-related decline in photosynthesis. There was little evidence that the age-related decline in photosynthesis was due to decreases in contents of N or Rubisco. The decrease in rates of photosynthesis from current-year to older needles was instead related to a twofold decrease in rates of photosynthesis per unit nitrogen and V _cmax/Rubisco (i.e., in vivo specific activity).     

Short-term changes in free radical scavengers and chloroplast pigments in Pinus canariensis needles as affected by mild drought stress

Potted Pinus canariensis seedlings were subjected to mild drought by withholding irrigation for one week. This treatment induced a reduction in maximum stomatal conductance (50 mmol m⁻² s⁻¹) compared to irrigated controls (130 mmol m⁻² s⁻¹). Needle water potentials of non-irrigated trees were maintained at control level (-0.44 MPa). Such a mild drought is a potential oxidative stressor due to the production of active oxygen species (AOS) in illuminated chloroplasts which lack CO₂ due to stomatal closure. Photoprotective pigments (e.g. the xanthophyll cycle) may avoid this situation through light energy dissipation, and antioxidants such as ascorbic acid, tocopherols, and glutathione, may detoxify AOS. Concentrations of ascorbate, glutathione, chlorophyll, and the xanthophyll cycle carotenoids were minimal in the evening (under low light) compared to light-saturated conditions. α-Carotene was highest in the evening. These short-term changes were not affected by drought. The xanthophyll cycle pool tended to be more de-epoxidized in strongly illuminated needles of non-irrigated trees at the beginning of the experiment, but this effect was transient. The glutathione pool was more oxidized in needles of non-irrigated trees (up to 20 percnt; of total vs. 10 percnt; at control) after the xanthophyll changes took place, whereas the redox state of ascorbate remained stable.     

Effect of tree age on needle morphology and anatomy of Pinus uliginosa and Pinus silvestris – species-specific character separation during ontogenesis

Differences in morphological and anatomical characters of needles between seedlings, saplings and adult trees of the endangered Pinus uliginosa from the Węgliniec Nature Reserve in SW Poland were examined biometrically and statistically assessed using the Student's t-test, Tukey–Kramer test, step-wise discrimination and agglomeration on Euclidean distances according to Ward's method. Pinus sylvestris adults and seedlings were used as comparative material. The results show that needles of all three P. uliginosa generations differ significantly from each other. In seedling needles, several anatomical characters were similar to those of P. sylvestris growing in the vicinity of the reserve. However, P. uliginosa had a lower number of resin canals, lower frequency of fibre-like sclerenchyma cells and higher frequency of thin-walled sclerenchyma cells with large lumens in the spaces between vascular bundles. Needle characters of saplings and adult trees of both species were distinctly more different than it was the case in the seedling stage.     

Physiological characteristics of Japanese red pine, Pinus densiflora Sieb. et Zucc., in declined forests at Mt. Gokurakuji in Hiroshima Prefecture, Japan

The decline of Japanese red pine trees (Pinus densiflora Sieb. et Zucc.) at Mt. Gokurakuji (693 m a.s.l.), 30 km west of Hiroshima city, west Japan, was studied. The effects of air pollution and acid deposition on the physiological characteristics of the trees, especially those of the needles, were investigated. Ozone concentration was not correlated with the physiological status of the needles and SO₂ concentration was not high in the declined area. NO₂ concentration correlated negatively with needle longevity while it correlated positively with ethylene emission from 1-year-old needles. Average needle longevity was about 2.8 years in non-declined areas; however the longevity was 1.3 years in the most polluted area. The minimal fluorescence at night (F ₀)of 1-year-old needles decreased with increasing NO₂ concentration. The maximum stomatal conductance (gl), net photosynthesis (P _n)and intercellular CO₂ concentration (C _i) in the declined areas were lower than in the non- declined areas (about 50%, 30% and 20% lower, respectively). The lower C _isuggested that the major part of the decrease in P _ncan be explained by stomatal restriction. The soil pH, N content and C/N ratio showed no significant difference between the declined and non-declined areas. The physiological disorders of needles were due to the damage by air pollutants, and important roles of NO₂ are suggested. Lowering of P _n and the shortening of needle longevity appear to be the main causes of the decline in pines in the forest decline area. Received: 16 December 1998 / Accepted: 7 January 2000     

Effects of Inoculation with PGPR Bacillus and Pisolithus tinctorius on Pinus pinea L. Growth,Bacterial rhizosphere Colonization,and Mycorrhizal Infection

The effect of co-inoculation with Pisolithus tinctorius and a PGPR belonging to the genus Bacillus (Bacillus licheniformis CECT 5106 and Bacillus pumilus CECT 5105) in enhancing growth of Pinus pinea plants and the changes that occurred in rhizosphere microbial communities and the degree of mycorrhization were evaluated. Both bacterial strains of Bacillus promote the growth of Pinus pinea seedlings, but this biological effect does not imply a synergic effect with mycorrhizal infection. However, the positive response to mycorrhiza in a longer-term experiment it could be expected. The introduction of both inocula causes an lateration in the microbial rhizosphere composition, despite the low levels of inocula that were found at the end of the assay.     

Control of Adventitious Root Production and Hypocotyl Hypertrophy of Sunflower (Helianthus annuus) in Response to Flooding

Needles of 20-year-old Scots pine trees (Pinus silvestris L.) were permitted to photoassimilate ¹⁴CO₂ for 1 h on different dates during the growing season. The loss of radioactivity from current, 1-year-old, and 2-year-old needles was followed, and the translocation of photoassimilated ¹⁴CO₂ from older needle age-classes to the elongating new needles studied. The effects of good mineral and water supply on translocation were also considered. In the spring, 1-year-old and 2-year-old needles accumulated ¹⁴C. These reserves, together with current photosynthate, were utilized when the trees started growing. The 1-year-old needles exported ¹⁴C to the current needles during the first weeks of elongation of the later, while no such translocation occurred from the 2-year-old needles. Removal of the 1-year-old needles resulted in translocation of assimilates from the 2-year-old needles to the current needles. The general pattern of translocation observed in the control trees was not changed when the trees were fertilized and irrigated. The new needles started to export assimilates in the middle of July when the photosynthetic rate per needle was comparable with that of the older age-classes. This occurred about 4 weeks after positive net photosynthesis was first measured for the current shoot. The current needles of trees with good nutrient and water supply seemed to become self-sufficient in photoassimilates earlier than the current needles of the control trees.     

Effects of <Emphasis Type="Italic">Pinus pinea</Emphasis> litter on seed germination and seedling performance of three Mediterranean shrub species

Pinus pinea L. is a coniferous tree inhabiting the Mediterranean Basin, which appears widely distributed in the Iberian Peninsula, frequently associated to Mediterranean shrub species, such as Cistus salvifolius L., C. libanotis L. and Halimium halimifolium (L.) Willk. (Cistaceae). This study investigated the effects (chemical and physical) of P. pinea needles on the seed germination and early growth of these three shrub species. Laboratory experiments were carried out in order to assess an expected negative effect by using different treatments to seeds (physical layer and two aqueous extracts concentrations). Seed germination of all species was inhibited when treated with aqueous extracts and the degree of inhibition was higher when using the extract with the highest concentration, while no effect was observed with the physical layer treatment. Seedling performance was also sensitive to the different treatments, showing the most stressed values with the high concentration extract. The results from this study highlight the importance of allelopathy of P. pinea needles as a mechanism for controlling seed germination and seedling performance of some Mediterranean shrub species.     

Variation with Tree Age of Needle Cuticle Topography and Stomatal Structure in Pinus radiate D. Don

Scanning electron microscopy reveals differences in the surfacetopography and stomatal structure of 1-year-old needles of Pinusradiata sampled from trees of different ages. The cuticularridges on young-tree needles show an even pitch, whereas theridges on mature-tree needles appear slightly puckered, withsmall discontinuities. The stomata on mature-tree needles havea smaller pore (10–15 µm) than young-tree needles(15–20 µm). In young trees a fine rodlet, or tubularwax covers the walls of the guard and subsidiary cells. Thestomatal antechamber predominating in mature-tree needles containsan amorphous wax, which frequently closes the pore between theoverarching stomatal lips. The yield of crude wax from chloroformextracts of needles of trees of all ages is approximately 0.2per cent, and there is more of the acidic component in the waxof mature-tree needles. It is suggested that wax occlusionswithin the stomatal antechamber of P. radiata may contributeto mature-tree resistance to the needle pathogen, Dothistromapini Hulbary.     

Incorporation of atmospheric 15NO2-nitrogen into free amino acids by Norway spruce Picea abies (L.) Karst.

During spring and autumn 1991, potted 6-yearold spruce trees (Picea abies (L.) Karst.) were fumigated with 60 nl·1^{–1 15}NO₂ for 4 days under controlled conditions in constant light. Current and previous flush needles, the bark and the fine roots were analysed for total ¹⁵N content and incorporation of ¹⁵N into the -amino nitrogen of free amino acids. In addition, in vitro nitrate reductase activity and stomatal conductance of the needles were measured. Nitrate reductase activity was significantly higher in the needles of fumigated trees compared to control trees exposed to filtered air. With an average of 9.1% ¹⁵N, free glutamate was the pool with the most label. Taking into account the time-course of the labelling of this pool, this figure can be taken as an estimate of the minimum contribution of NO₂ to the N nutrition of the needles. ¹⁵N-labelled amino acids were also detected in the bark and the roots, indicating export from the needles.     

The toxicity of antibiotics and herbicides on in vitro adventitious shoot formation of Pinus pinea L. cotyledons

Summary The toxicity of three antibiotics commonly used to eliminate Agrobacterium tumefaciens from plant tissue during transformation were tested to determine their effect on Pinus pinea L. morphogenesis. Cefotaxime and vancomycin at a dose of 250 μg ml⁻¹, as well as ticarcillin at 300 μg ml⁻¹, were essentially nontoxic to the culture and significantly enhanced regeneration and shoot development. Kanamycin, a widely used selection agent for plant transformation, strongly inhibited regeneration even at very low doses. On the contrary, phosphinothricin and its ammonium salt glufosinate in the commercial formulation Finale? proved to be the best selection agents because they were less toxic to stone pine cotyledons. Thus, they could be applied immediately after transformation to avoid escapes. Other schemes for selection and recovery of transgenic stone pine trees are discussed.     

Pine volatiles mediate host selection for oviposition by Thaumetopoea pityocampa (Lep., Notodontidae)

The oviposition behaviour and host selection by females of the pine processionary moth, Thaumetopoea pityocampa, was studied under two conditions: in a field insectary and in a pine stand. An oviposition choice test was conducted in an insectary cage, using artificial Christmas trees (ACTs) baited with extracts from four pine species: Pinus pinea, Pinus pinaster, Pinus halepensis and Pinus brutia, plus a control. Females oviposited significantly more egg batches on the ACT baited with a P. brutia extract, while no oviposition occurred on the control ACT. In a large P. pinea stand, two groups of randomly selected trees were marked and baited respectively with an extract of P. brutia, which was the preferred species under insectary conditions and with a solvent, to act as control. Results showed that oviposition in the field followed an aggregated pattern, fitting a negative binomial distribution and that trees baited with P. brutia extracts, received a significantly larger number of egg‐batches than control trees. In parallel, the volatiles emitted by all pine extracts tested where analysed by gas chromatography and mass spectrometry (GC‐MS), coupled to a time‐of‐flight (TOF) mass analyser after solid phase microextraction (SPME), resulting in the identification of 26 compounds. Comparative chromatograms showed qualitative differences among the pine species used, some compounds being present in one of them only. Findings demonstrate for the first time that: (i) T. pityocampa females discriminate among bouquets extracted from different host pine species and exhibit oviposition preferences; and (ii) olfactory cues play an important role in mediating the selection process.     

Effects of an enhanced ultraviolet-B irradiation on photosynthetic apparatus of several forest coniferous tree species from different locations

The effect of UV-B on the photosynthetic apparatus of coniferous trees: Picea abies (L.) Karst., Picea pungens (Engelm.), Pinus sylvestris (L.), Pinus cembra (L.) and Abies alba (Mill.) was investigated. Three and four-year-old plantlets coming from different latitudes, longitudes and altitudes were used. The experiment was carried out in greenhouse. Two doses of ultraviolet-B irradiation were applied: control=0, low dose=11.32 and high dose=22.64 kJ·m⁻²·d⁻¹ UV-B_BE (biologically effective irradiance of UV-B). Measurements of chlorophyll fluorescence, gas exchange, chlorophyll and flavonoids content were carried out. Response of forest trees to an increased UV-B radiation depends on species, location of place of pantalets collecting and UV-B dose. Pinus cembra, Picea abies and Pinus sylvestris from high altitude (1000 m a.s.l.) were less sensitive to UV-B than these from plain location. The altitude determined adaptation of forest coniferous trees to an enhanced UV-B radiation much more than the latitudinal gradient. Permanent discoloration was observed only on the young needles of the fir plantlets that were grown in light limiting conditions. Photosynthetic parameters were affected by the UV-B radiation. Both maximal and the steady state fluorescence of chlorophyll were reduced as a consequence of elevated UV-B in case of some species. The chlorophyll content was enhanced, increased or was not affected according to species and to locations. The flavonoids content in the needles increased with chlorophyll content at both UV-B treatments. An opposite trend was found in the control. The increased content of screening pigments in the needles of all the tested coniferous trees was detected. Picea abies and Picea pungens photosynthesis response curves to the light and to the intercellular CO₂ concentration did not change significantly under increased UV-B because of higher concentration in screening pigments in leaves. The increased concentration of flavonoids in forest litter may lead to changes in the biogeochemical cycle in the forest ecosystem.     

The ion concentration of dew condensed on Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.) needles

Summary Dew droplets collected with pipettes from coniferous needles were analysed for their ionic composition. Almost all samples of dew taken from Scots pine trees (Pinus sylvestris) showed significantly higher ion concentrations than those taken from Norway spruce trees (Picea abies). This can be explained by the micromorphology of the needle surface. The higher microscale roughness of the wax layer of a pine needle causes a more efficient flux of atmospheric aerosol particles compared to the spruce needle surface. Dew on coniferous needles is shown to be capable of maintaining pH values below 3 for several hours.     

Does nitrogen availability control rates of litter decomposition in forests?

The effects of increased exogenous N availability on rates of litter decomposition were assessed in several field fertilization trials. In a jack pine (Pinus banksiana Lamb.) forest, needle litter decomposed at the same rate in control plots and in plots fertilized with urea and ammonium nitrate (1350 kg N ha^-1) with or without P and K. Mixed needle litter of western hemlock (Tsuga heterophylla (Raf.) Sarg.), western red cedar (Thuja plicata Donn) and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) incubated in plots recently amended with sewage sludge (500 kg N ha^-1) lost less weight during 3 years than did litter in control plots. Forest floor material also decomposed more slowly in plots amended with sewage sludge. Paper birch (Betula papyrifera Marsh.) leaf litter placed on sewage sludge (1000 kg N ha^-1), pulp sludge, or sewage-pulp sludge mixtures decomposed at the same rate as leaf litter in control plots. These experiments demonstrate little effect of exogenous N availability on rates of litter decomposition.The influence of endogenous N availability on rates of litter decomposition was examined in a microcosm experiment. Lodgepole pine (Pinus contorta var. latifolia Engelm.) needle litter collected from N-fertilized trees (525 kg N ha^-1 in ammonium nitrate) were 5 times richer in N than needles from control trees (1.56% N versus 0.33% N in control trees), but decomposed at the same rate. Green needles from fertilized trees contained twice as much N as needles from control trees (1.91% N versus 0.88% N), but decomposed at the same rate. These experiments suggest that N availability alone, either exogenous or endogenous, does not control rates of litter decomposition. Increased N availability, through fertilization or deposition, in the absence of changes in vegetation composition, will not alter rates of litter decomposition in forests.     

Anatomical changes with needle length are correlated with leaf structural and physiological traits across five Pinus species

The genus Pinus has wide geographical range and includes species that are the most economically valued among forest trees worldwide. Pine needle length varies greatly among species, but the effects of needle length on anatomy, function, and coordination and trade‐offs among traits are poorly understood. We examined variation in leaf morphological, anatomical, mechanical, chemical, and physiological characteristics among five southern pine species: Pinus echinata, Pinus elliottii, Pinus palustris, Pinus taeda, and Pinus virginiana. We found that increasing needle length contributed to a trade‐off between the relative fractions of support versus photosynthetic tissue (mesophyll) across species. From the shortest (7 cm) to the longest (36 cm) needles, mechanical tissue fraction increased by 50%, whereas needle dry density decreased by 21%, revealing multiple adjustments to a greater need for mechanical support in longer needles. We also found a fourfold increase in leaf hydraulic conductance over the range of needle length across species, associated with weaker upward trends in stomatal conductance and photosynthetic capacity. Our results suggest that the leaf size strongly influences their anatomical traits, which, in turn, are reflected in leaf mechanical support and physiological capacity.     

Non-linear response of stomata in Pinus koraiensis to tree age and elevation

Knowledge on variations in stomata is useful in reflecting leaf physiological characteristics of CO₂ uptake and water transpiration, and predicting the responses of plants to future climate change. Stomatal density and number of stomatal rows (current-year, 1- and 2-year-old needles) in relation to tree age (ranging from 25 to 320 years old), elevation (ranging from 738 to 1,380 m a.s.l.), and sun exposure (sun and shade exposure) were investigated in Pinus koraiensis trees. Stomatal density and number of stomatal rows in relation to tree age and elevation showed a humped curve with the maximum values at intermediate levels of tree age (210 years old) and elevation (1,050 m a.s.l.), respectively. Needle age but not sun exposure significantly affected the stomatal density across tree ages and elevations. Our results suggest that variations in stomatal density of Pinus koraiensis needles are related to ontogenetic growth and environmental factors.