Global application of stable hydrogen and oxygen isotopes to wildlife forensics

Stable isotopes are being increasingly used in wildlife forensics as means of determining the origin and movement of animals. The heavy isotope content of precipitated water and snow (D_p, ¹⁸O_p) varies widely and systematically across the globe, providing a label that is incorporated through diet into animal tissue. As a result, these isotopes are potentially ideal tracers of geographic origin. The hydrogen and oxygen isotope tracer method has excellent potential where (1) spatial variation of precipitation isotopes exist, and (2) strong, mechanistic relationships link precipitation and isotope ratios in biological tissue. Here, we present a method for interpolation of precipitation isotope values and use it to create global basemaps of growing-season (GS) and mean annual (MA) D_p and ¹⁸O_p. The use of these maps for forensic application is demonstrated using previously published isotope data for bird feathers (D_f) in North America and Europe. The precipitation maps show that the greatest potential for applying hydrogen and oxygen isotope forensics exists in mid- to high-latitude continental regions, where strong spatial isotope gradients exist. We demonstrate that D_f/D_p relationships have significant predictive power both in North America and Europe, and show how zones of confidence for the assignment of origin can be described using these predictive relationships. Our analysis focuses on wildlife forensics, but the maps and approaches presented here will be equally applicable to criminal forensic studies involving biological materials. These maps are available in GIS format at .     

Linking biodiversity to ecosystem function: implications for conservation ecology

We evaluate the empirical and theoretical support for the hypothesis that a large proportion of native species richness is required to maximize ecosystem stability and sustain function. This assessment is important for conservation strategies because sustenance of ecosystem functions has been used as an argument for the conservation of species. If ecosystem functions are sustained at relatively low species richness, then arguing for the conservation of ecosystem function, no matter how important in its own right, does not strongly argue for the conservation of species. Additionally, for this to be a strong conservation argument the link between species diversity and ecosystem functions of value to the human community must be clear. We review the empirical literature to quantify the support for two hypotheses: (1) species richness is positively correlated with ecosystem function, and (2) ecosystem functions do not saturate at low species richness relative to the observed or experimental diversity. Few empirical studies demonstrate improved function at high levels of species richness. Second, we analyze recent theoretical models in order to estimate the level of species richness required to maintain ecosystem function. Again we find that, within a single trophic level, most mathematical models predict saturation of ecosystem function at a low proportion of local species richness. We also analyze a theoretical model linking species number to ecosystem stability. This model predicts that species richness beyond the first few species does not typically increase ecosystem stability. One reason that high species richness may not contribute significantly to function or stability is that most communities are characterized by strong dominance such that a few species provide the vast majority of the community biomass. Rapid turnover of species may rescue the concept that diversity leads to maximum function and stability. The role of turnover in ecosystem function and stability has not been investigated. Despite the recent rush to embrace the linkage between biodiversity and ecosystem function, we find little support for the hypothesis that there is a strong dependence of ecosystem function on the full complement of diversity within sites. Given this observation, the conservation community should take a cautious view of endorsing this linkage as a model to promote conservation goals. Received: 2 September 1999 / Accepted: 26 October 1999     

Escape of volcanic gas into shallow groundwater systems at Unzen Volcano (Japan): evidence from chemical and stable carbon isotope compositions of dissolved inorganic carbon

 Chemical and stable carbon isotopic analyses of dissolved inorganic carbon (DIC) were carried out for groundwater samples collected from cold springs and shallow wells in the Unzen volcanic region in 1999 and 2000. All of the data sets plotted on the carbon isotope ratio (δ¹³C) vs 1/DIC diagram can be explained by mixing of volcanic CO₂ with DIC equilibrated with soil CO₂. Groundwater DIC showing a high mixing ratio of volcanic CO₂ appears to have a tendency to distribute along two major faults near the activity center of the 1990–1995 eruption. This suggests that these faults are escape routes of volcanic CO₂ diffused into the volcanic edifice. The total flux of the volcanic DIC discharged from the cold springs is shown to be one to two orders of magnitude lower than the roughly estimated flux of volcanic CO₂ discharged from the summit during the eruptive period. Received: November 10, 2001 / Accepted: June 6, 2002 Acknowledgments The Unzen Scientific Drilling Project, Ministry of Education, Culture, Sports, Science and Technology (Japan), provided funding. We acknowledge G. Lyon and W. Gooley for stable carbon isotope measurement, K. Amita for DIC analysis, and students of Kyoto University and Okayama University of Science for assistance in field work. Correspondence to:S. Ohsawa     

The use of stable isotopes to partition evapotranspiration fluxes into evaporation and transpiration

It is crucial to partition evapotranspiration (ET) into evaporation (E) and transpiration (T) components for better understanding eco-hydrological processes and their underlying mechanisms, and improving the establishment and validation of hydrological models at the ecosystem scale. Traditional eddy covariance technique serves as a useful tool to estimate ET, but it encounters difficulties in quantifying the relative contribution of E and T to ET. Combining with eddy covariance technique, it is possible to partition ET based on the measurements of stable oxygen and hydrogen isotopes in liquid and vapor phases of water in the Soil–Plant–Atmosphere Continuum (SPAC) system. The key challenge is to precisely determine the oxygen-18 and deuterium isotopic compositions of ET (δ_ET), E (δ_E) and T (δ_T). δ_E can be estimated based on the Craig–Gordon model. δ_T is usually approximated by the δ¹⁸O and δD of water in xylem or twig (δx), assuming δ_T equals δx under isotopic steady state (SSA). However, the SSA is only likely satisfied during midday in field conditions. The diurnal variations of δ_T is affected by isotopic composition of atmospheric water vapor (δ_v) and leaf water at the evaporating sites (δ_L,e), and relative humidity, resulting in the non-steady-state behavior of δ_T at the sub-daily cycles. δ_ET can be estimated using the flux-gradient approach or the Keeling plot by measuring the vapor mixing ratio and δ_v at different heights in the surface layer. However, δ_v observations by the traditional cold trap/mass spectrometer method are limited to a coarse time resolution, leading to discrete time series of δ_ET. It is now possible to make in situ and high time resolution measurements of δ_v and to analyze a large number of plant and soil samples due to technical and instrumental advances in recent years. It provides an opportunity to improve the model prediction of δ_L,e, and more importantly, to calculate δ_T from δ_L,e without invoking the SSA. Combining with the flux-gradient approach or the Keeling plot technique, continuous δ_ET measurements can be made. It offers us a premise for accurate ET partitioning on diurnal time scale. In this review we introduced the recent advances, foci and challenges for studies on ET partitioning using the stable isotopes technique.     

The photosynthetic drought physiology of a diverse group of southern hemisphere conifer species is correlated with minimum seasonal rainfall

1. The instantaneous and integrated leaf gas exchange of 13 species of southern hemisphere conifers grown under identical glasshouse conditions were examined to determine whether there was any correlation between the characteristics of water use at the leaf level and environmental water availability.
2. In the conifer species examined, the minimum ratio of internal to ambient CO₂ measured in leaves during artificially imposed drought [( c _i/ c _a)_min] was strongly correlated with the minimum rainfall observed within the natural range of each species. This suggests that the distributions of these species are constrained by the drought tolerance of their photosynthetic apparatus.
3. A good correlation was found between the ratio of internal to ambient CO₂ measured in leaves under optimal conditions ( c _i/ c _a)_max and leaf δ¹³C (and hence inferred ∫[ c _i/ c _a]). Neither of these, however, correlated with the environmental parameters considered most likely to be limiting species distribution, i.e. precipitation and altitude.
4. These data suggest that decreasing water availability may have been the major factor responsible for the restriction and extinction of conifers in the southern hemisphere.     

The use of stable isotopes to partition evapotranspiration fluxes into evaporation and transpiration

It is crucial to partition evapotranspiration (ET) into evaporation (E) and transpiration (T) components for better understanding eco-hydrological processes and their underlying mechanisms, and improving the establishment and validation of hydrological models at the ecosystem scale. Traditional eddy covariance technique serves as a useful tool to estimate ET, but it encounters difficulties in quantifying the relative contribution of E and T to ET. Combining with eddy covariance technique, it is possible to partition ET based on the measurements of stable oxygen and hydrogen isotopes in liquid and vapor phases of water in the Soil–Plant–Atmosphere Continuum (SPAC) system. The key challenge is to precisely determine the oxygen-18 and deuterium isotopic compositions of ET (δ_ET), E (δ_E) and T (δ_T). δ_E can be estimated based on the Craig–Gordon model. δ_T is usually approximated by the δ¹⁸O and δD of water in xylem or twig (δx), assuming δ_T equals δx under isotopic steady state (SSA). However, the SSA is only likely satisfied during midday in field conditions. The diurnal variations of δ_T is affected by isotopic composition of atmospheric water vapor (δ_v) and leaf water at the evaporating sites (δ_L,e), and relative humidity, resulting in the non-steady-state behavior of δ_T at the sub-daily cycles. δ_ET can be estimated using the flux-gradient approach or the Keeling plot by measuring the vapor mixing ratio and δ_v at different heights in the surface layer. However, δ_v observations by the traditional cold trap/mass spectrometer method are limited to a coarse time resolution, leading to discrete time series of δ_ET. It is now possible to make in situ and high time resolution measurements of δ_v and to analyze a large number of plant and soil samples due to technical and instrumental advances in recent years. It provides an opportunity to improve the model prediction of δ_L,e, and more importantly, to calculate δ_T from δ_L,e without invoking the SSA. Combining with the flux-gradient approach or the Keeling plot technique, continuous δ_ET measurements can be made. It offers us a premise for accurate ET partitioning on diurnal time scale. In this review we introduced the recent advances, foci and challenges for studies on ET partitioning using the stable isotopes technique.     

Discontinuous gas exchange in dung beetles: patterns and ecological implications

This study correlates a distinctive pattern of external gas exchange, referred to as the discontinuous gas exchange cycle (DGC), observed in the laboratory, with habitat associations of five species of telecoprid dung beetles. The beetles were chosen from a variety of habitats that would be expected to present different amounts of water stress. All five species exhibited DGC. Sisyphus fasciculatus has been recorded only in woodland areas, and does not have strict spiracular control during its DGC. Anachalcos convexus and Scarabaeus rusticus are associated with open mesic habitats. Both species exhibit a distinct DGC, previously found in some other insect species, but intermediate within this study group. Sc. flavicornis and Circellium bacchus are typically found in arid regions, and have the most unusual form of DGC, with spiracular fluttering during the burst phase. These results support the hypothesis that spiracular fluttering reduces respiratory water loss. From this study we conclude that the DGC is an ancestral adaptation, most probably as a result of anoxic environments in underground burrows, but that spiracular control is enhanced to reduce respiratory water loss in beetle species that live in arid habitats. Received 4 August 1999 / Accepted: 7 October 1999     

Response of leaf surfaces and gas exchange to wind stress and acid mist in birch (Betula pubescens)

 This research demonstrates that a leaf’s response to acid mist is dependent on the integrity of the leaf cuticle and that significant differences in the structural and physiological disturbances in leaves can be attributable to different types of wind action. Betula pubescens Ehrh. plants were located at adjacent, but contrasting, sites to create different wind treatments: (i) direct wind action, (ii) indirect wind action and (iii) shelter from wind action (control). In combination with the wind treatments, acidic (pHs 5 and 3) or neutral (pH 7) mists were applied weekly. Wind action significantly increased visible leaf injury, microscopic cuticular lesions and cuticular conductance (g _c ), but reduced photosynthetic rate (P _N ) and stomatal conductance (g _s ) compared to shelter. Wind action combined with acid mist was more injurious than wind action alone, but leaves sheltered from wind action were highly resistant to the damaging effects of acid mist. Direct wind action combined with pH 3 mist resulted in the highest values of g _c and the greatest number of cuticular lesions. By contrast, indirect wind action combined with pH 3 mist induced most visible injury, but relatively low values of g _c and few microscopic cuticular lesions. Acid mist reduced P _N only when leaves had been damaged by wind action. Higher values of g _c were associated both with increases in the area of visible leaf injury and with the number of cuticular lesions. Compensatory increase in P _N of healthy tissue was evident in leaves exposed to combinations of wind action and acid mist. Received: 10 November 1997 / Accepted: 6 March 1998     

Assessing the impact of elevated O3 and CO2 on gas exchange characteristics of differently K supplied clonal Norway spruce trees during exposure and the following season

 Well-supplied and K-deficient 4-year-old clonal Norway spruce trees were exposed to combinations of two levels of ozone (20 and 80 nl l^{–  1} O₃) and carbon dioxide (350 and 750 μl l^{–  1} CO₂) to study the effects of possible future climate factors on gas exchange characteristics. The fumigation was performed in environmental chambers for a complete growing season. After the exposure, plants were cultivated outdoors to investigate possible recovery and delayed effects. During the exposure 1-year-old needles responded to the 80 nl l^{–  1} O₃ treatment by a sharp but transient decrease of both apparent carboxylation efficiency (CE) and maximum photosynthetic capacity (A₂₅₀₀). Elevated CO₂ also reduced CE and A₂₅₀₀. The effect became stronger in the course of the exposure and was accompanied by decreases of N and P as well as chlorophyll contents. In case of K deficiency, the acclimation response of current-year needles was even more pronounced reflecting lower sink capacities for carbon metabolites. The joint application of elevated O₃ and CO₂ resulted in the lowest values of gas exchange parameters and chlorophyll contents. At the beginning of the growing season after the exposure and under outdoor conditions, all these treatment effects disappeared in the needles which had developed during the fumigation. In the course of the development of the new flush, however, the well-supplied 1-year-old needles which had been treated with 80 nl l^{–  1} O₃ and 350 μl l^{–  1} CO₂ in the year before, exhibited a sharp decline of CE and A₂₅₀₀. Simultaneously, chlorotic mottle and bands developed. These delayed symptoms are discussed in the context of the previously published “memory” effect for O₃ (Sandermann et al. 1989). Additionally, evidence is presented that shoot development is altered in plants which had been exposed to elevated O₃. Accepted: 5 August 1996     

Response of net ecosystem gas exchange to a simulated precipitation pulse in a semi-arid grassland: the role of native versus non-native grasses and soil texture

Physiological activity and structural dynamics in arid and semi-arid ecosystems are driven by discrete inputs or pulses of growing season precipitation. Here we describe the short-term dynamics of ecosystem physiology in experimental stands of native (Heteropogon contortus) and invasive (Eragrostis lehmanniana) grasses to an irrigation pulse across two geomorphic surfaces with distinctly different soils: a Pleistocene-aged surface with high clay content in a strongly horizonated soil, and a Holocene-aged surface with low clay content in homogenously structured soils. We evaluated whole-ecosystem and leaf-level CO₂ and H₂O exchange, soil CO₂ efflux, along with plant and soil water status to understand potential constraints on whole-ecosystem carbon exchange during the initiation of the summer monsoon season. Prior to the irrigation pulse, both invasive and native grasses had less negative pre-dawn water potentials ( _pd), greater leaf photosynthetic rates (A _net) and stomatal conductance (g _s), and greater rates of net ecosystem carbon exchange (NEE) on the Pleistocene surface than on the Holocene. Twenty-four hours following the experimental application of a 39 mm irrigation pulse, soil CO₂ efflux increased leading to all plots losing CO₂ to the atmosphere over the course of a day. Invasive species stands had greater evapotranspiration rates (ET) immediately following the precipitation pulse than did native stands, while maximum instantaneous NEE increased for both species and surfaces at roughly the same rate. The differential ET patterns through time were correlated with an earlier decline in NEE in the invasive species as compared to the native species plots. Plots with invasive species accumulated between 5% and 33% of the carbon that plots with the native species accumulated over the 15-day pulse period. Taken together, these results indicate that system CO₂ efflux (both the physical displacement of soil CO₂ by water along with plant and microbial respiration) strongly controls whole-ecosystem carbon exchange during precipitation pulses. Since CO₂ and H₂O loss to the atmosphere was partially driven by species effects on soil microclimate, understanding the mechanistic relationships between the soil characteristics, plant ecophysiological responses, and canopy structural dynamics will be important for understanding the effects of shifting precipitation and vegetation patterns in semi-arid environments.     

Relation of water transport to leaf gas exchange properties in three mangrove species

Mangrove species more tolerant to salinity may function with less efficient water transport, which may be related to more conservative water use. To test the hypothesis, we investigate the gas exchange and hydraulic properties of three mangrove species: Rhizophora mangle L., Laguncularia racemosa Gaert and Avicennia germinans (L.)L. Experiments were performed with adult plants growing naturally in the field under a salinity of 35‰. Gas exchange parameters showed that A. germinans had significantly higher photosynthetic rates, and lower stomatal conductance and transpiration rates, compared to the other two mangroves. In concert with this, instantaneous water use efficiency was significantly high in A. germinans, intermediate in L. racemosa and lowest in R. mangle. The hydraulic parameters of the three mangrove species were in the lowest end of the range reported for tropical trees. However, the three mangrove species exhibited measurable differences in hydraulic parameters related to the control of water requirements for maintenance of carbon gain. L. racemosa and A. germinans showed less efficient water transport at shoot level but were the more efficient species in water use at the leaf level in comparison to R. mangle. Received: 7 April 1999 / Accepted: 25 July 1999     

Direct assessment of water exchange on a Gd(III) chelate bound to a protein

 The Gd(III) complex of 4-pentylbicyclo[2.2.2]octane-1-carboxyl-di-l-aspartyl-lysine-derived DTPA, [GdL(H₂O)]^2–, binds to serum albumin in vivo, through hydrophobic interaction. A variable temperature ¹⁷O NMR, EPR, and Nuclear Magnetic Relaxation Dispersion (NMRD) study resulted in a water exchange rate of k ²⁹⁸ _ex=4.2×10⁶ s^–1, and let us conclude that the GdL complex is identical to [Gd(DTPA)(H₂O)]^2– in respect to water exchange and electronic relaxation. The effect of albumin binding on the water exchange rate has been directly evaluated by ¹⁷O NMR. Contrary to expectations, the water exchange rate on GdL does not decrease considerably when bound to bovine serum albumin (BSA); the lowest limit can be given as k _{ex, GdL-BSA}=k _ex, GdL / 2. In the knowledge of the water exchange rate for the BSA-bound GdL complex, the analysis of its NMRD profile at 35  °C yielded a rotational correlation time of 1.0 ns, one order of magnitude shorter than that of the whole protein. This value is supported by the longitudinal ¹⁷O relaxation rates. This indicates a remarkable internal flexibility, probably due to the relatively large distance between the protein- and metal-binding moieties of the ligand. Received: 25 June 1998 / Accepted: 11 August 1998     

Modeling dynamics of stable carbon isotopic exchange between a boreal forest ecosystem and the atmosphere

Stable isotopes of CO₂ contain unique information on the biological and physical processes that exchange CO₂ between terrestrial ecosystems and the atmosphere. In this study, we developed an integrated modeling system to simulate dynamics of stable carbon isotope of CO₂, as well as moisture, energy, and momentum, between a boreal forest ecosystem and the atmosphere, as well as their transport/mixing processes through the convective boundary layer (CBL), using remotely sensed surface parameters to characterize the surface heterogeneity. It has the following characteristics: (i) it accounts for the influences of the CBL turbulent mixing and entrainment of the air aloft; (ii) it scales individual leaf‐level photosynthetic discrimination up to the whole canopy (Δ_canopy) through the separation of sunlit and shaded leaf groups; (iii) it has the capacity to examine the detailed interrelationships among plant water‐use efficiency, isotope discrimination, and vapor pressure deficit; and (iv) it has the potential to investigate how an ecosystem discriminates against ¹³C at various time and spatial scales. The monthly mean isotopic signatures of ecosystem respiration (i.e. δ¹³C_R) used for isotope flux calculation are retrieved from the nighttime flask data from the intensive campaigns (1998–2000) at 20 m level on Fraserdale tower, and the data from the growing season in 1999 are used for model validation. Both the simulated CO₂ mixing ratio and δ¹³C of CO₂ at the 20 m level agreed with the measurements well in different phases of the growing season. On a diurnal basis, the greatest photosynthetic discrimination at canopy level (i.e. Δ_canopy) occurred early morning and late afternoon with a varying range of 10–26‰. The diurnal variability of Δ_canopy was also associated with the phases of growing season and meteorological variables. The annual mean Δ_canopy in 1999 was computed to be 19.58‰. The monthly averages of Δ_canopy varied between 18.55‰ and 20.84‰ with a seasonal peak during the middle growing season. Because of the strong opposing influences of respired and photosynthetic fluxes on forest air (both CO₂ and ¹³CO₂) on both the diurnal and seasonal time scales, CO₂ was consistently enriched with the heavier ¹³C isotope (less negative δ¹³C) from July to October and depleted during the remaining months, whereas on a diurnal basis, CO₂ was enriched with the heavier ¹³C in the late afternoon and depleted in early morning. For the year 1999, the model results reveal that the boreal ecosystem in the vicinity of Fraserdale tower was a small sink with net uptake of 29.07 g ¹²C m^?2 yr^?1 and 0.34 g ¹³C m^?2 yr^?1.     

The use of RAPD analysis to classify Triticum accessions

 Crop germplasm collections contain a considerable percentage of misclassified accessions which may affect the use of germplasm for agricultural crop improvement. The objective of this study was to determine if random amplified polymorphic DNA (RAPD) analysis could be used to reclassify misclassified Triticum accessions. Twelve accessions suspected to be misclassified, based on morphological characters, as either macha or vavilovii wheat were studied using RAPD and cytological analyses. In the RAPD analysis, a dendrogram, based on Jaccard genetic similarity coefficients, grouped 5 dicoccum-like, 1 timopheevii-like, and 6 monococcum-like accessions with Triticum dicoccum, T. timopheevii, and T. monococcum accessions, respectively. These results were confirmed by the cytological analysis. A RAPD marker specific to the D genome was also detected. This study suggests that RAPD analysis can be used to classify germplasm and to distinguish some species in Triticum. Received: 12 June 1998 / Accepted: 18 August 1998     

The use of stable isotopes in the study of human pathophysiology

The growing prevalence of metabolic diseases including fatty liver disease and Type 2 diabetes has increased the emphasis on understanding metabolism at the mechanistic level and how it is perturbed in disease. Metabolomics is a continually expanding field that seeks to measure metabolites in biological systems during a physiological stimulus or a genetic alteration. Typically, metabolomics studies provide total pool sizes of metabolites rather than dynamic flux measurements. More recently there has been a resurgence in approaches that use stable isotopes (e.g. ²H and ¹³C) for the unambiguous tracking of individual atoms through compartmentalised metabolic networks in humans to determine underlying mechanisms. This is known as metabolic flux analysis and enables the capture of a dynamic picture of the metabolome and its interactions with the genome and proteome. In this review, we describe current approaches using stable isotope labelling in the field of metabolomics and provide examples of studies that led to an improved understanding of glucose, fatty acid and amino acid metabolism in humans, particularly in relation to metabolic disease. Examples include the use of stable isotopes of glucose to study tumour bioenergetics as well as brain metabolism during traumatic brain injury. Lipid tracers have also been used to measure non-esterified fatty acid production whilst amino acid tracers have been used to study the rate of protein digestion on whole body postprandial protein metabolism. In addition, we illustrate the use of stable isotopes for measuring flux in human physiology by providing examples of breath tests to measure insulin resistance and gastric emptying rates.     

Trends in plant carbon concentration and plant demand for N throughout this century

 Atmospheric CO₂ concentration has increased by 25% over the preindustrial level. A parallel increase in C concentration and decreases in N concentration and δ¹³C of plants grown throughout this century have been observed in plant specimens stored in herbaria. We tested our previous results in a study of 12 more species collected in the western Mediterranean throughout this century (1920–1930, 1945–1955, and 1985–1990) and tree rings of Quercus pubescens from the same area. These changes were accompanied by apparent increases in condensed tannin concentration. A decreasing trend in δ¹⁵N both in herbarium material and tree rings was also found, indicating that ecosystems might cope with higher plant N demand by decreasing N losses and increasing N fixation and mineralization. These results may contribute to a better understanding of the effects of global change on carbon and nitrogen cycling. Received: 12 November 1995 / Accepted: 17 May 1996     

Growth and gas exchange responses of Hevea brasiliensis seedlings to inoculation with Glomus mosseae

 The beneficial effect of arbuscular mycorrhizal (AM) fungi on plant growth is well known, but the physiological processes involved are still discussed. The purpose of this study was to determine if Glomus mosseae affects the growth of Hevea brasiliensis seedlings and, if it is the case, if it could be associated with variations in leaf CO₂ and H₂O gas exchange. H. brasiliensis rubber trees were grown for 9 months in a medium containing either propagules of G. mosseae or sterilized inoculum. Plant size, root collar diameter and leaf area, as well as net CO₂ assimilation, stomatal conductance (g_s) and water-use efficiency of photosynthesis were evaluated during the first 5 stages of growth. At stage 2, a growth depression occurred in the mycorrhizal seedlings coincident with the first AM infections. Then, at stage 5, Glomus mosseae-inoculated plants had moderate colonization (47% of root length) and were taller than control plants with a larger root collar diameter and an enhanced leaf organogenesis. This enhanced growth was accompanied by increased photosynthesis, transpiration, and stomatal conductance. After 9 months, dry weights of shoots and roots of inoculated plants were greater than those of controls by 27 and 17%, respectively. Received: 10 May 1997 / Accepted: 9 September 1997     

Effects of ozone and mild drought stress on gas exchange, antioxidants and chloroplast pigments in current-year needles of young Norway spruce [Picea abies (L.) Karst.]

 To investigate the effects of ozone exposure and soil drought, singly and in combination, on gas exchange, antioxidant contents and pigments in current-year needles of Norway spruce [Picea abies (L.) Karst.] 4-year-old seedlings were fumigated in growth chambers with either charcoal-filtered air or with 100 nl l^–1 ozone for 106 days. After 3 weeks a 20% reduction in gas exchange was observed in ozone-treated seedlings. However, no further decrease occurred in spite of continued ozone exposure. Whole needle ascorbate and apoplastic ascorbate increased until the end of the experiment and contents were 62% and 82%, respectively, higher than in ozone-free controls. This increase in ascorbate might have protected net photosynthesis from further decline. Ozone pre-treated plants and ozone-free controls were subjected to soil drought for 38 days which caused stomatal narrowing. Thereby ozone uptake was reduced when compared to well watered seedlings. At the end of the experiment drought alone, and even more in combination with ozone, had also caused an increase in ascorbate. Glutathione increased only in drought-stressed seedlings. The redox states of the ascorbate and the glutathione pools were not affected by any treatment. Superoxide dismutase activity declined under both stresses but was most reduced by ozone alone. While chlorophyll and neoxanthin contents remained unchanged, carotenes were significantly decreased upon drought. The combination of O₃ and drought induced increased lutein contents, an increased pool size of the xanthophyll cycle as well as an increased epoxidation status of the xanthophyll cycle. These results suggest that spruce needles seem to be able to acclimate to ozone stress but also to drought stress by increasing their ascorbate pools and protecting pigments. Received: 15 September 1997 / Accepted: 24 March 1998     

The impact of elevated CO2 on growth and photosynthesis in Agrostis canina L. ssp. monteluccii adapted to contrasting atmospheric CO2 concentrations

 The aim of this study was to characterise growth and photosynthetic capacity in plants adapted to long-term contrasting atmospheric CO₂ concentrations (C _a). Seeds of Agrostis canina L. ssp. monteluccii were collected from a natural CO₂ transect in central-western Italy and plants grown in controlled environment chambers at both ambient and elevated CO₂ (350 and 700 μmol mol⁻¹) in nutrient-rich soil. Seasonal mean C _a at the source of the plant material ranged from 610 to 451 μmol CO₂ mol⁻¹, derived from C₄ leaf stable carbon isotope discrimination (δ¹³C). Under chamber conditions, CO₂ enrichment stimulated the growth of all populations. However, plants originating from elevated C _a exhibited higher initial relative growth rates (RGRs) irrespective of chamber CO₂ concentrations and a positive relationship was found between RGR and C _a at the seed source. Seed weight was positively correlated with C _a, but differences in seed weight were found to explain no more than 34% of the variation in RGRs at elevated CO₂. Longer-term experiments (over 98 days) on two populations originating from the extremes of the transect (451 and 610 μmol CO₂ mol⁻¹) indicated that differences in growth between populations were maintained when plants were grown at both 350 and 700 μmol CO₂ mol⁻¹. Analysis of leaf material revealed an increase in the cell wall fraction (CWF) in plants grown at elevated CO₂, with plants originating from high C _a exhibiting constitutively lower levels but a variable response in terms of the degree of lignification. In vivo gas exchange measurements revealed no significant differences in light and CO₂ saturated rates of photosynthesis and carboxylation efficiency between populations or with CO₂ treatment. Moreover, SDS-PAGE/ LISA quantification of leaf ribulose bisphosphate carboxylase/oxygenase (Rubisco) showed no difference in Rubisco content between populations or CO₂ treatments. These findings suggest that long-term adaptation to growth at elevated CO₂ may be associated with a potential for increased growth, but this does not appear to be linked with differences in the intrinsic capacity for photosynthesis. Received: 16 August 1996 / Accepted: 19 October 1996     

Water relations and gas exchange in Fagus sylvatica L. and Quercus petraea (Mattuschka) Liebl. in a mixed stand at their southern limit of distribution in Europe

Water status and gas exchange of beech (Fagus sylvatica L.) and sessile oak [Quercus petraea (Mattuschka) Liebl.] were studied in a mixed stand in the Montejo de la Sierra forest (central Iberian Peninsula), one of the southernmost locations of both species in Europe. Gas exchange and water potential were measured in leaves at different canopy levels over several days in two growing seasons. The daily variation pattern was established with the measurements of three selected dates per year, representative of the soil moisture content situations in early, mid- and late summer. A similar daily time course of leaf water potential was found for the two species. Nevertheless, beech showed a most noticeable decrease of water potential at midmorning and maintained lower leaf water potential than oak in the early afternoon. In 1994 the sessile oak saplings showed higher values of predawn water potential (Ψ_pd) than beech at the end of summer, when soil moisture content was lowest (20 cm depth). Beech showed a significantly lower net assimilation rate (A) than sessile oak for leaves under the same PPFD. Maximum net photosynthesis values (A _max) for beech and sessile oak on sunny leaves were 10.1±0.4 μmol m^–2 s^–1 and 17.8±1.7 μmol m^–2 s^–1 respectively, and those for water vapour stomatal conductance (g _wv) were 265±31 mmol m^–2 s^–1 and 438±74 mmol m^–2 s^–1. Differences in A and g _wv between the two species were maintained throughout the day on all measurement dates. No clear relationship was found between water status of saplings and stomata performance; there was only a negative correlation between Ψ_pd and g _wvmid in beech. Nevertheless, a significant response to the air vapour pressure gradient between leaf and air was translated into stomata closure on an hourly basis, more intensively in beech. Received: 4 March 1999 / Accepted: 21 December 1999