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Isoprenoid emissions of Quercus spp. (Q. suber and Q. ilex) in mixed stands contrasting in interspecific genetic introgression Blackwell Publishing, Ltd.
Michael Staudt, Céline Mir, Richard Joffre, Serge Rambal, Aurélie Bonin, Damien Landais and Roselyne Lumaret CNRS Centre d’Ecologie Fonctionnelle et Evolutive, 1919 route de Mende, 34293 Montpellier cedex 5, France
Summary Author for correspondence: Michael Staudt Tel: +33 4 67 61 32 72 Fax: +33 4 67 41 21 38 Email:
[email protected] Received: 20 February 2004 Accepted: 28 April 2004
• Among oak species, Quercus ilex is classified as a monoterpene emitter and Q. suber is mainly known as a nonisoprenoid emitter. The extent and origin of this diversification is unknown. • We examined intra- and interspecific emission variability in two mixed stands which differed in their level of hybridization and reciprocal genetic introgression based on variations in cytoplasmic (chloroplast DNA) and nuclear (allozyme) markers. • At both sites all trees identified as Q. ilex, or as recent descendants from Q. ilex × Q. suber hybrids, emitted monoterpenes. Of Q. suber trees (genetically introgressed or not by Q. ilex), 91% were also monoterpene emitters, and the remainder nonemitters. One tree identified as a Q. canariensis × Q. ilex hybrid emitted both isoprene and monoterpenes. Compared with Q. ilex, the standard emission rate of Q. suber was higher in summer and lower in autumn. Both species emitted the same monoterpenes, proportions of which showed significant intra- and interspecific variability. • The results suggest that Q. suber populations in the French Mediterranean intrinsically emit monoterpenes, and that gene flow between oak species contributes to diversification of emission signatures. Key words: basal emission rate, chemotaxonomy, chemotypes, evergreen oaks, genetic introgression, isoprene, monoterpenes, VOC (volatile organic compound) emissions. New Phytologist (2004) 163: 573–584 © New Phytologist (2004) doi: 10.1111/j.1469-8137.2004.01140.x
Introduction Emissions of volatile organic compounds (VOC) from terrestrial vegetation are of considerable interest to atmospheric scientists because they interact in air pollution events and affect climate by influencing the atmosphere’s oxidative capacity and by contributing to particle formation and growth (Kesselmeier & Staudt, 1999; Monson & Holland, 2001; Sharkey & Yeh, 2001). Air chemistry and climate models need large-scale emission inventories that estimate the annual load of phytogenic VOCs into the atmosphere on regional and global scales. These estimations are highly uncertain, largely because of our limited knowledge of the genetic diversity of emission sources. Oaks have been recognized as among the greatest VOC emitters (Harley et al., 1999). Inside the chloroplasts their
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leaves produce large amounts of volatile isoprenoids in a lightand temperature-dependent process. At 30°C the emission typically consumes 1–2% of assimilated carbon, but the portion can be considerably increased under drought and hot-weather conditions (Sharkey & Loreto, 1993; Bertin & Staudt, 1996; Staudt & Bertin, 1998). The physiological role of this type of VOC production is still an enigma. There is increasing evidence that it protects the photosynthetic apparatus against high-temperature damage by enhancing membrane stability, or against oxidative stresses by scavenging oxidants such as ozone (Loreto & Velikova, 2001; Sharkey et al., 2001; Peñuelas & Llusià, 2002). It may also act as a redox valve, preventing the phosphate limitations of photosynthesis (Logan et al., 2000). The genus Quercus (oaks) includes > 300 woody species. It is widespread in the northern hemisphere, where it represents
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the dominant vegetation of temperate forests and shrublands (Nixon, 1993). Almost all the investigated species of oak were found to produce the hemiterpene isoprene, as many other terrestrial plants do. Interestingly, some Eurasian species, in particular Mediterranean oaks, show diversification in VOC production (Loreto et al., 1998; Csiky & Seufert, 1999). Quercus ilex (Holm oak) and Quercus suber (Cork oak), two widespread evergreen oaks in the western Mediterranean region, have been reported to be, respectively, a monoterpene emitter and a nonemitter instead of isoprene emitters (Kesselmeier & Staudt, 1999; Loreto, 2002). The assignment of Q. suber as a nonemitting species relies on data from measurements made on a few individuals in Italy (Steinbrecher et al., 1997; Delfine et al., 2000). Yet an earlier study in Portugal (Pio et al., 1993), as well as a recent study by Owen et al. (2001), report strong monoterpene emissions from this species. This discrepancy indicates the existence of geographic variability in the overall capacity to emit volatiles, which may be related to past genetic isolation of populations, adaptations to local growth conditions, or hybridization between emitting and nonemitting oak species. Quercus ilex and Q. suber are not very close genetically and belong to two distinct clades (Manos et al., 1999; Belahbib et al., 2001; Toumi & Lumaret, 2001; Bellarosa et al., 2004), for example (according to Schwarz, 1937) to the subsections Sclerophyllodrys (Schwarz) and Cerris (Spach), respectively. However, recent studies based on interspecific diagnostic genetic markers (mainly chloroplast DNA and allozymes) have pointed out nuclear and cytoplasmic genome exchanges caused by hybridization between Q. suber and Q. ilex in some geographic areas, particularly in the eastern and southern parts of Morocco and the eastern part of the Iberian Peninsula (Toumi & Lumaret, 1998; Belahbib et al., 2001; Lumaret et al., 2002; Castro et al., 2003). There have been no attempts to investigate the intraspecific variability of emissions in Q. suber, as is the case for many other species. For Q. ilex some emission studies were conducted on populations of several dozen individuals: these were either adult trees, to explore emission variability in natural forest; or potted trees, to study emission responses to environmental conditions (Staudt et al., 2001a, 2001b, 2003). In all populations, distinct differences in emission patterns were observed among individuals. Emission signatures of individuals were highly preserved with respect to leaf age and leaf crown position, and were independent of treatments that changed emission rates by more than one order of magnitude. These results provide clear evidence of genetic variability at population level. Further investigation is needed to understand the extent and origins of isoprenoid emission diversification in Quercus and other taxa, and to improve the accuracy of large-scale emission inventories. Furthermore, it offers the opportunity to use emission signatures as chemotaxonomic markers to identify subspecies and to define provenances, as has been done with plant extracts in numerous monoterpene-storing plant species (Lewinsohn et al., 1993).
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In the present study, we investigated for the first time the intraspecific variability of isoprenoid emissions from Q. suber, and expanded our previous study on Q. ilex to include more western and eastern populations in France (Staudt et al., 2001a). We characterized isoprenoid emissions of Cork oaks and Holm oaks growing in two mixed stands in sites located in two French regions which had been shown to differ substantially in their level of interspecific genetic exchanges between the two species. One region (Catalonia) was characterized by total, and probably ancient, cytoplasmic introgression of Q. suber by the Q. ilex genome and, on the basis of nuclear diagnostic markers, by a substantial proportion of individuals from more recent hybrid origin. The other region (Provence) showed a very reduced level of cytoplasmic interspecific exchange and limited nuclear gene flow occurring locally (Toumi & Lumaret, 1998; Lumaret, 2003; Lumaret et al., 2003). The main objectives are: to (i) analyse and compare intra- and interspecific quantitative and qualitative variation for VOC emissions; and (ii) identify the putative effect of genetic exchanges between the two oak species by assuming that higher interspecific exchanges may result in higher intraspecific variation for VOC production in each species.
Materials and Methods Plant material A total of 90 trees were studied in two sites of southern France (Fig. 1). Site one, Mas Anglade, is located in French Catalonia in the eastern hills of the Pyrenean Mountains (42°29′19′′ N, 02°51′39′′ E, elevation 300 m) close to the Spanish border. In that region Quercus ilex L. can be considered the native and predominant tree species. However, many woodlands have been managed for cork production for at least two centuries. In the measuring site, approx. 80% of the trees are Quercus suber L., 15% Q. ilex and 5% other species such as the white
Fig. 1 Location of the two study sites in southern France. The Quercus ilex populations investigated in a previous study (Staudt et al., 2001a) lie about midway, close to the northern point of the Gulf of Lion.
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oak Quercus pubescens Willd. Approximately two-thirds of the trees studied are located on the plateau and western slope of the site, whereas the other third are growing in a small valley. Twenty-one adult trees with the morphological appearance of Q. ilex (or close to it, see below) and 21 adult trees with the appearance of Q. suber were labelled and collected at that site. Site two is located in Port-Cros island (43°00′06′′ N, 06°22′36′′ E, elevation 0 m), a national park in Provence region. Port-Cros is part of the Hyères islands which lie approx. 10 km offshore of the southernmost point of the French Riviera. In Port-Cros approximately half the trees were located inland as understorey trees in forests dominated by Aleppo pine (Pinus halepensis Mill.), and the other half in the coastal vegetation of the island’s western seashore. At that site, which did not show any evidence of management, 25 adult trees morphologically looking Q. ilex, 22 adult trees with the morphological appearance of Q. suber, and one juvenile tree (approx. 10–15 yr old), showing leaf traits between evergreen and deciduous oaks, were analysed. Genetic characterization of individual trees In a first step, individuals were assigned to Q. ilex or Q. suber on the basis of several morphological traits according to species identification in Flora Europaea (Tutin et al., 1993), but primarily according to the cortical production of cork. However, as shown from experimental crosses, first-generation hybrid individuals have no cork (Natividade, 1936). Even for most other characters these individuals are usually closer morphologically to Q. ilex, and cannot be easily distinguished in natural conditions (Lumaret et al., 2002). Moreover, in natural interspecific hybridization, on the basis of variation in cytoplasmic DNA (cpDNA), which is maternally inherited in oaks (Dumolin et al., 1995), Q. ilex was found to be predominantly (but not exclusively) the mother species (Lumaret et al., 2002). Initial hybridization events were usually followed by backcrosses with either parental species, Q. suber being predominantly the pollen-bearing species. Consequently, cytoplasmic introgression was observed to occur mainly from Q. ilex into Q. suber and to be maintained over very long periods in large areas (Belahbib et al., 2001). Conversely, morphologically intermediate individuals between Q. ilex and Q. suber are rare, even in mixed stands. This suggests that the hybrids may have to cope with selective disadvantage, and that most of the initial unidirectional or bidirectional nuclear genetic introgression (including the genes coding for morphological traits) which may have occurred between the two oak species disappears with successive backcrosses, more particularly if these involve the same parental species. Therefore first-generation hybrids are expected to combine alleles specific to each parental species at each nuclear diagnostic locus, and the individuals, which correspond morphologically to one species but still possess nuclear alleles specific to the other, probably derive from a relatively recent
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interspecific hybridization event. Conversely, trees belonging morphologically to one species but which show the cpDNA molecular characteristics of the other parental species, without evidence of nuclear introgression, can be considered to derive from a more ancient hybridization event. In the present study, each individual tree collected for VOC emission analysis was characterized genetically for cytoplasmic and nuclear markers, namely restriction fragment-length polymorphism (RFLP) of cpDNA, which corresponds to very distinct molecular lineages in Q. ilex and Q. suber (Belahbib et al., 2001; Petit et al., 2003); allozyme variation at three nuclear loci located on distinct chromosomes and coding for phosphoglucose isomerases (EC 5.3.1.9), leucine aminopeptidases (EC 3.4.11.1) and acid phosphatases (EC 3.1.3.2). All these markers had been shown to be diagnostic between the two oak species, and the respective analytical techniques have been described previously (Toumi & Lumaret, 1998, 2001; Lumaret et al., 2002). In addition, using the same methodology and appropriate specific enzyme markers (Toumi & Lumaret, 2001), the putative hybrid origin was tested of a juvenile tree growing in Port-Cros and observed to be morphologically intermediate between an evergreen oak species and a deciduous oak species. Measurements of isoprenoid emissions, leaf gas exchange and morphological traits Measurements of isoprenoid emissions, leaf gas exchange (photosynthesis, transpiration), and structural traits were made on cut branches sampled during two campaigns, one in autumn (end October 2001, 85 trees) and one in summer ( June 2002, 90 trees). Samples were usually taken from the upper sun-exposed parts of the tree crown by means of 9 m telescopic pole pruners. However, during winter 2001/02 three Q. ilex trees in the Catalonian site were chopped down by the farmers, so the summer measurements of these trees had to be made on leaves of resprouts. Weather conditions in the summer campaign were good and stable throughout all sampling days at both sites. In the autumn campaign they were variable: During sampling days weather conditions were balmy and sunny at the Catalonian site, while cool and stormy on the island of Port-Cros. Sampled branches were immediately submerged in water and re-cut underwater to avoid xylem air embolism. They were transported in water to the Institute and stored either in a glasshouse (autumn campaign) or outdoors in a half-shaded place (summer campaign) until all samples were assayed for isoprenoid emissions. Emission assays lasted for 3–9 d, during which water was regularly replaced. After measurements, leaves were harvested to determine their fresh weight (microbalance Mettler PM200, Mettler-Toledo SA, Virofley, France), projected area (Delta-T Area Meter MK2, Delta-T Devices Ltd, Cambridge, UK), and dry weight after drying for at least 48 h in a ventilated oven at 60°C. These data were used to calculate specific leaf mass (SLM, leaf dry mass per unit projected leaf
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area in g m−2) and relative leaf water content (LWC, %) here defined as: (fresh weight − dry weight)/fresh weight × 100. Foliar isoprenoid emissions and gas exchange were determined indoors by clamping one or several terminal leaves of a sample in a temperature and light-controlled dynamic flowthrough gas-exchange chamber located in a nearby laboratory. The custom-made chamber (volume 100 ml) consists of a transparent lid and a water jacket and Teflon-coated metal housing equipped with two thermocouples to measure leaf and air temperature, and a quantum sensor (Licor, PAR-SB 190, Lincoln, NE, USA) to measure photosynthetic photon flux density (PPFD). Leaf gas exchange (photosynthesis, transpiration) was measured by drawing a constant portion of the inlet and outlet air through a CI-301 infrared CO2 gas analyser (CID Inc., Camas, WA, USA) via tubes enclosing two humidity sensors (HIH-3602C, Honeywell Inc., IL, USA). Data were collected by a 21X datalogger (Campbell Scientific Ltd, Shepsherd, UK). The net airflow rate inside the chamber was 0.32 mmol s−1. Measurements were made at a constant leaf temperature of 30 ± 1°C and PPFD of approx. 900 µmol m−2 s−1 after an acclimation time of at least 1 h under the same conditions. The humidity of the inlet air was set to approx. 50% by bypassing a portion of the air stream through a humidifier. Several trees were repeatedly assayed in a campaign because leaves of their samples showed low physiological activity. When photosynthesis was lower than approx. 3 µmol m−2 s−1, leaves of the same branch were re-assayed during the following days, and in case activity remained low a new branch was sampled and re-assayed under the same conditions. Generally, in summer almost all branches sampled showed immediate normal photosynthetic activity, whereas in autumn many samples, in particular those of Q. suber, became active only after some days. Samples of some trees in the autumn remained completely inactive. The data for these measurements were not considered in the data evaluation because they do not allow conclusions on the tree’s capacity to emit isoprenoids. Isoprenoid emissions were measured by drawing cuvette exhaust air (1 l at 0.1 l min−1) through a glass tube containing 200 mg of either Tenax, for monoterpenes and higher terpenes or Carbotrap, for isoprene. These traps were analysed by capillary gas chromatography (Chrompack CP9003 with flame ionization detector, Varian SA, Les Ulis, France) after thermal desorption (10 min at 200°C) and preconcentration on a capillary cold trap (−100°C for monoterpenes, −150°C for isoprene) by a TCT 4002 thermodesorber (Chrompack). Complete resolution of the monoterpenes emitted was achieved with a Sil 8CB fused silica capillary column (25 m × 0.32 mm, 1.2 µm df, Chrompack) using the following multiramp temperature program: 4 min at 65°C, 2.5°C min−1 to 80°C, 2°C min−1 to 100°C, 10°C min−1 to 220°C. Isoprene emission was analysed according to Van Eijk & Kotzias (1994) using a fused silica PLOT Al2O3/KCl column (25 m × 0.32 mm, Chrompack). The temperature program was 2 min at 80°C, 8°C min−1 to 200°C, 10 min at 200°C.
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Carrier gas was helium (1.2 ml min−1) in all cases. Both precision and accuracy were within 2–5% as determined by repeated measurements of standards (Fluka Chemie AG, Buchs, Switzerland; Roth, Karlsruhe, Germany) at realistic concentrations. The lower detection limit of the analytical system is approx. 0.5 p.p.b. (v/v) for the monoterpenes. Data analysis Data for the summer and autumn campaigns were treated separately. For each data set, one-way and two-way s ( 3.0, SPSS Inc., Chicago, IL, USA) were used to test differences among genotypes, species and geographical origin (site), and interactions. F tests were used to determine the statistical significance (P < 0.05), and the Student– Newman–Keuls test for the multiple pair-wise comparisons. Kruskal–Wallis s were applied to ranked data if tests for normality or equal variance failed. In a first step, we tested whether Q. ilex and Q. suber morphotypes, identified by nuclear genetic markers to be of recent hybrid origin between Q. ilex and Q. suber (see below), differed from ‘pure’ individuals (trees with no evidence of recent hybrid origin) in their leaf emission, gas exchange or anatomical features. In both sites and for both campaigns, statistically significant differences were seen only between the two species (individuals from recent hybridization or not), but not between ‘pure’ individuals and individuals from recent hybrid origin within a species (one-way , P > 0.05). Because of the absence of significant differences in emission and of measured variables, data for individuals derived from recent hybrids were pooled to the respective data sets for the ‘pure’ trees and analysed for differences with respect to the factors species and site (two-way s). Additional t tests were applied to evaluate the effects of growth conditions (shaded vs open vegetation) within a species and site, and differences between emitting and nonemitting Q. suber trees. Pearson’s product moment correlation or, in case residuals showed no normal distribution (determined by the Kolmogorov–Smirnov test), Spearman’s rank order correlation (all 3.0) were used to detect correlations between measured variables. Cluster analysis was carried out on the fraction of individual monoterpenes using Ward’s technique with the Euclidian distance measure (R-GUI Software, GNU Operating System Free Software Foundation, www.gnu.org/). χ2 analysis of contingency tables ( 3.0) was used to compare frequencies of emission types (main clusters) between populations.
Results Individual genetic characterization All Q. suber and Holm individuals from Catalonia (Mas Anglade) possessed a cpDNA haplotype belonging to the Q. ilex lineage,
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Research Table 1 Number of trees from Port-Cros (Provence) and Mas Anglade (Catalonia) showing either a Quercus ilex or a Quercus suber morphotype and possessing a cpDNA molecule and/or alleles at one, two or three diagnostic allozyme loci characteristic of the opposite species Allozyme loci Site Provence Catalonia
Species (morphotype)
cpDNA
1
2
3
Q. ilex (25) Q. suber (22) Q. ilex (21) Q. suber (21)
1 1 0 21
1 1 7 3
2 0 1 1
3 0 0 0
site did so only at a single locus. This indicates that the hybridization events from which these individuals derived are more recent in the Provence site than in the Catalonian site. Generally, most individuals of recent hybrid origin had leaf morphology close to Q. ilex, and no cortical cork. One apparently different tree on Port-Cros was identified as a hybrid between Q. ilex and Quercus canariensis Willd., an isopreneemitting deciduous oak species introduced in Port-Cros approx. 60 yr ago (Roux & Thinon, 1987). On the basis of cpDNA variation analysed in that hybrid, Q. ilex was identified as the mother parental species. Intra- and interspecific emission variability
indicating a complete cytoplasmic introgression of Q. suber by Q. ilex in that site (Table 1). In the Provence site (Port-Cros), all trees analysed possessed a cpDNA haplotype corresponding to their respective morphology, except one Q. ilex tree and one Q. suber tree, for which the cpDNA of the opposite species was observed. On the basis of nuclear genetic markers, 19 trees, seven in Provence (15% of the trees sampled in that site) and 12 in Catalonia (29%) combined alleles of both species at least at one locus, and were therefore identified as of recent hybrid origin (Table 1). Among these, most individuals in the Provence site possessed alleles of the opposite species at two or three loci, whereas most individuals in the Catalonian
None of the Q. suber and Q. ilex trees studied emitted significant amounts of isoprene, except the Q. ilex × Q. canariensis hybrid which emitted isoprene and monoterpenes in proportions of approx. 60/40% (Fig. 2). Four Q. suber trees in the Catalonian site did not emit isoprene or monoterpenes in significant amounts, although their leaves were physiologically fully active during both campaigns (Fig. 2, trees on extreme right). These trees were considered as nonemitters. Foliage of all other trees emitted the same principal monoterpenes: α-pinene, β-pinene, sabinene, myrcene and limonene (Fig. 2b). In both species and sites, the fraction of individual compounds (percentage of total emission) varied among individual trees.
Fig. 2 Monoterpene emissions from 90 individual Quercus ilex and Quercus suber trees growing in two mixed populations located in Provence and French Catalonia. (a) Emission rate of the sum of the five main compounds measured under standard conditions, once in summer and once in autumn; (b) mean emission composition (± SE) of these two measurements. Four Q. suber individuals in the Catalonian site did not emit volatile isoprenoids in significant amounts (shown at the extreme right hand of the figure). The tree at the extreme left (IC) corresponds to a hybrid individual between Q. ilex and Quercus canariensis. All other trees were found to emit exclusively monoterpenes (sorted according to the limonene fraction in each category). Individuals marked with an asterisk are trees with the morphological appearance of Q. ilex and Q. suber derived from recent hybridization between these two species (Table 1). §, Measurements in the summer campaign were made on leaves of young resprouts because trees were chopped down between the two campaigns.
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For a given tree the emission profile was almost stable. Commonly the limonene portions decreased by 2–9% from the autumn to the summer campaign, while the percentage of other main compounds slightly increased. Several other monoterpenes were found in traces but were close to the detection limit and therefore discarded from further data evaluation. Mean emission rates of the sum of the five main compounds determined at standard conditions were significantly different between Q. ilex and Q. suber in both sites and during both campaigns. In autumn the emission rates of Q. ilex in the Provence and Catalonia sites were 4.7 and 2.8 times higher, respectively, than those of Q. suber collected at the same sites (Table 2). In autumn there was also a significant difference between the Provence and Catalonia sites: the emission rates of Provence samples collected at the beginning of November, during a cold and rainy period, were substantially lower than those of the Catalonia samples, collected at end of October under mild, sunny weather conditions (Table 2). The lowered emission rates of the Port-Cros samples were also paralleled by lowered rates of photosynthesis, although differences in photosynthesis were not significant. In the summer campaign weather conditions were stable and no site differences were seen in emission or photosynthesis rates. Standard emission rates were much higher than in the autumn, especially those of Q. suber. Unlike what was observed in the autumn, in summer leaves of Q. suber emitted significantly more monoterpenes than Q. ilex leaves on a leaf-area basis, and particularly on a leaf dry-matter basis. In both campaigns mean fractions of four monoterpenes were significantly different between the two species (Table 2), except the limonene fractions which showed strong intraspecific variations. Differences in mean fractions were more pronounced in the Provence site than in the Catalonian site, and a site effect was detected for the α-pinene, sabinene and myrcene fractions in emissions from Q. ilex during summer (species–site interaction, P < 0.05). Cluster analysis of the emission signatures (Fig. 3) split trees into four main types, frequencies of which differ significantly among populations in some cases (Table 3). First, the pinene type which emitted mainly α-, β-pinene plus sabinene – this was the most frequent type (72% of Q. ilex and 51% of Q. suber) and was particularly common in the Q. ilex population at the Provence site (80% of trees). Second, a rather rare type emitting high limonene was observed exclusively in the Q. suber population of the Provence site, with exception of one Q. ilex tree derived from recent interspecific hybridization at the Catalonian site. Third, a more common limonene type which emitted limonene in intermediate proportions – this type was present in all populations but was more frequent in the Catalonian Q. suber population (53% vs 20–28% in the other populations). The final type emitted myrcene in high proportions and was represented by a sole Q. ilex tree at the Catalonian site. Within the main clusters, several subclusters appeared that separate the two oak species well, except for two and four
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Fig. 3 Dendrogram resulting from cluster analysis of the monoterpene-emission signatures measured during summer on 85 Quercus ilex (I) and Quercus suber (S) trees in two mixed populations in Catalonia (C) and Provence (P). Beside a single tree emitting mainly myrcene, three main clusters can be distinguished: a pinene type emitting mainly α- and β-pinene (53 trees), and two limonene types (31 trees) emitting limonene in either high (> 80%) or intermediate (30 –50%) portions. Trees marked with an asterisk are individuals derived from recent hybridization between Q. ilex and Q. suber.
trees in the pinene and limonene clusters, respectively (Fig. 3). More particularly, in the pinene-type cluster one Q. ilex and one Q. suber, both from Provence, were classified with trees of the opposite species. In the limonene-type clusters a Catalonian tree that showed Q. ilex morphology (or very close to it) and possessed one and two alleles peculiar to Q. suber at two and one diagnostic enzyme loci, respectively (i.e. not a firstgeneration hybrid but an individual probably derived from recent interspecific hybridization), was classified in the
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Table 2 Leaf monoterpene emissions, photosynthesis and anatomical features of Quercus ilex and Quercus suber trees growing in mixed stands in two sites, one in Provence (Port-Cros) and one in French Catalonia (Mas Anglade) Emission rate
Site and species Autumn (no. of trees)* Q. ilex Provence (19) Q. ilex Catalonia (19) Q. suber Provence (18) Q. suber Catalonia (16)
Emission composition (sum = 100%)
nmol m−2 s−1
µg g−1 h−1
α-Pinene
β-Pinene
Sabinene
Myrcene
Limonene
Photosynth (µmol m−2 s−1)
SLM (g m−2)
LWC (%)
Leaf form (length/width)
2.1 ± 0.5 8.7 ± 0.5 0.5 ± 0.5 3.9 ± 0.5
4.8 ± 1.0 18.6 ± 1.0 1.3 ± 1.0 10.0 ± 1.1
33.8 ± 2.6 29.0 ± 2.6 24.6 ± 2.7 28.2 ± 2.9
21.3 ± 1.9 21.5 ± 1.9 14.9 ± 1.9 16.8 ± 2.0
17.6 ± 2.1 11.0 ± 2.1 24.4 ± 2.1 23.1 ± 2.3
6.8 ± 1.8 12.0 ± 2.8 3.0 ± 1.8 3.4 ± 1.9
20.6 ± 5.7 26.5 ± 5.7 33.1 ± 5.9 28.5 ± 6.2
5.5 ± 0.9 7.7 ± 0.9 4.4 ± 0.9 6.4 ± 0.9
217 ± 5 230 ± 5 164 ± 5 189 ± 6
44.8 ± 0.5 43.0 ± 0.5 45.9 ± 0.5 46.5 ± 0.5
2.3 ± 0.1 2.3 ± 0.1 1.9 ± 0.1 1.8 ± 0.1
ANOVA P values Species Site Species × site
< 0.001 < 0.001 0.762
< 0.001 < 0.001 0.801
0.010 0.891 0.188
0.002 0.678 0.809
< 0.001 0.074 0.225
< 0.001 0.189 0.795
0.064 0.347 0.611
0.084 0.050 0.609
< 0.001 < 0.001 0.227
