High chemodiversity in the structural and enantiomeric composition of volatiles emitted by Kermes oak populations in Southern France

Terrestrial vegetation is the major source of biogenic volatile organic compounds (BVOCs) affecting chemical and physical properties of the troposphere (Guenther et al., 2012; Glasius and Goldstein, 2016). BVOCs influence the oxidative capacity of the atmosphere and thereby the lifetime of greenhouse gases and are involved in the formation and growth of secondary organic aerosols. Large efforts have been made to characterize the BVOC emission capacity of numerous plant species and vegetation covers to estimate their fluxes in regional and global emission inventories. By contrast, the extent of the intraspecific emission variability, that is, the inherent differences in the emissions from individuals of a same species, remains largely unexplored, casting some doubt on the accuracy of emission inventories and the associated modeling of air chemistry processes. The C₅-isoprenoid isoprene is considered to be the most emitted BVOC. Isoprene is released from leaves in relevant rates of about 30% of the vascular plants being screened for isoprene emissions (Kesselmeier and Staudt, 1999; Fineschi et al., 2013) and accounts alone for about half of the global total BVOC emissions (ca. 500 10¹⁵ g a⁻¹; Guenther et al., 2012). Monoterpenes (MTs, C₁₀-isoprenoids) are estimated to be the second most emitted BVOCs with ca. 100 10¹⁵ g a⁻¹ globally and can dominate the total BVOC release of some vegetation types such as coniferous forests. The rates of isoprene and MTs emitted by a leaf are constantly modulated at different timescales by climatic and edaphic factors such as temperature and water availability (Peñuelas and Staudt, 2010) and by endogenic factors such as phenology and circadian rhythms (Liebelt et al., 2019). This makes the detection of intraspecific differences in emission quantities beyond absence/presence challenging (e.g., see Steinbrecher et al., 2013), especially if individuals of different populations cannot be grown and assayed under common environmental conditions (e.g., see van Meeningen et al., 2016). Yet, MTs exists in numerous isomers and MT producing plant species usually emit more than one of them. This allows detecting chemical polymorphism in MT producers based on compositional variations rather than on quantitative variations of single compounds. Many studies have characterized chemotypes in the populations of coniferous and aromatic plant species, which have sometimes been used as markers of provenances and/or ecotypes, that is, individuals that differ in their relationship to abiotic and biotic factors (e.g., Kännaste et al., 2013; Clancy et al., 2016; Junker-Frohn et al., 2019). In fact, MTs are often part of a specific biologically active mix of plant volatiles that can mediate vital interactions with heterotrophic organisms such as pollinating insects (Proffit et al., 2020). Knowledge on the composition of MT fluxes from vegetation also matters in air pollution and climate research, since individual MTs can differ considerably in their reactivity with atmospheric oxidants and the nature and yield of the reaction products (e.g., Kemper Pacheco et al., 2014; Junker-Frohn et al., 2019). In plants, MTs are synthesized from a wide range of terpene synthases, many of which produce more than 1 MT in distinct proportions (Degenhardt et al., 2009; Christianson, 2017). The product pattern typically includes several structural isomers, that is, MTs that differ in the basic configuration of their carbon skeletons and the position of double bounds. In addition, MTs in which a carbon atom is bonded to 4 different groups have mirror image forms (chiral forms), called stereoisomers or enantiomers (hereinafter denoted by (+) and (−)). Biochemical studies including analyses of stereoisomers observed that multiproduct enzymes typically produce either (+)- or (−)-enantiomers but rarely both. For example, in thyme 2 multiproduct enzymes have been identified producing a similar spectrum of 16 MTs but with opposite stereospecificity (Krause et al., 2013). Olfactory receptors distinguish enantiomers, which hence can exert different biological activities in the receiving organisms (Andersson, 2012; Zannoni et al., 2020b). Instead in the atmosphere, enantiomers exhibit identical gas-phase reactivity toward atmospheric oxidants (Leppla et al., 2023 and references therein). However, the stereo-specificity of their reaction products can influence the surface properties of particles on which they condense, which in turn may affect their cloud droplet formation potential (Bellcross et al., 2021; Gasso and Knobelspiesse, 2022). Several studies monitoring MT enantiomers in ambient air observed that the ratios of the 2 enantiomers of α- and β-pinene were not stable but varied spatially between and within forest ecosystems (Williams et al., 2007; Zannoni et al., 2020a) or temporally with daytime and season (Song et al., 2012; Yassaa et al., 2012). Given the identical physicochemical properties of enantiomers, these variations were interpreted as indicating changes of the predominant MT emission sources or sinks in the forest ecosystems, for example as shifts in the fractions of pinene emissions stemming from storage and de-novo-synthesis pools. Consistent with this interpretation, emission measurements of different compartments of a maritime pine plantation revealed that pinene emissions from foliage were consistently more enriched in (+)-enantiomers than the emissions from the stem surfaces (Staudt et al., 2019). More recently, Byron et al. (2022) monitored terpene concentrations in the air of an artificial tropical rainforest macrocosm during a drought cycle combined with ¹³CO₂ pulse-labeling and observed that the de-novo-synthesis of specific terpene enantiomers increased during certain phases of the cycle. In the same context, Song et al. (2014) investigated the light and temperature responses of pinene enantiomers emitted from the foliage of 3 Mediterranean plant species. That study found no evidence that in BVOC storing plants a specific pinene enantiomer stems from de-novo-synthesis as it was shown for ocimene and linalool in pine emissions (Staudt et al., 2017). Instead, Song and coworkers found evidence that the individuals of the same species can inherently differ in the enantiomeric fingerprints they emit. The presence of intraspecific chiral chemotypes could explain spatial variations of enantiomeric MT ratios in air over vegetation covers with similar floral composition.

With more than 400 species, oaks (Quercus) are among the most diversified and most abundant trees in temperate and subboreal forests of the Northern hemisphere (Kremer and Hipp, 2020). The vast majority of oaks are strong isoprene emitters (Csiky and Seufert, 1999; Kesselmeier and Staudt, 1999; Monson et al., 2012; Bao et al., 2023 and references therein). However, several nonemitting species or species that emit MTs at high rates have been identified in the Quercus subgenus Cerris (Hipp et al., 2020). Some of them are widespread in the Western Mediterranean basin, namely Q. ilex, Q. suber, and Q. coccifera. Yet, earlier emission studies on these species, which usually examined only a few individuals, sometimes reported quite divergent results. For example, field studies on Q. ilex trees in Italy reported that pinene was the main compound emitted (Bertin et al., 1997), whereas in Spain it was limonene (Peñuelas and Llusià, 1999). Even more strikingly, Q. suber was classified as a non-emitting oak species by Steinbrecher et al. (1997), whereas Pio et al. (1993) described it as a strong MT emitter. These apparent inconsistencies pointed to an unknown endogenous factor determining their emissions. Indeed, later studies focusing on the intraspecific emission variability in Q. ilex and Q. suber populations revealed individuals emitting distinctly different MT profiles, and more rarely the existence of nonemitting and dual isoprene-MT emitting individuals (Staudt et al., 2001; Staudt et al., 2004; Loreto et al., 2009; Welter et al., 2012). Interestingly, there were similar chemotypes in all populations of these 2 oak species, indicating the existence of a common diversification mechanism. So far, no study has specifically addressed the intraspecific variability of the emissions from Q. coccifera (QC, Kermes Oak). Nevertheless, previous observations suggest that chemotypes also exist in this species. Thus, in a population in Northeast Spain, limonene was the major emitted compound from Q. coccifera (Llusia et al., 2013), whereas α-pinene dominated in a population in Southern France (Ormeno et al., 2009; Olivier et al., 2011). Further, in a study examining the emission responses of Q. coccifera to light and temperature, Staudt and Lhoutellier (2011) noted persistent divergent emission profiles among individuals. In the present study, we report the results of an emission screening performed on 80 seed-grown QC saplings originating from 4 populations along a northeast–southwest transect in Southern France. Specifically, we address the following questions:

• Do foliar isoprenoid emissions of QC individuals intrinsically differ in the quality or quantity of VOCs emitted and how do these compare to those reported for other MT emitting oak species?

• Does intraspecific variability exist only in the occurrence of structural isomers or also in the enantiomeric composition?

• Does the occurrence of chemotypes vary among populations and is this variation associated with local climate conditions and leaf ecophysiological traits?

QC is an evergreen shrub or small tree native to the Mediterranean basin (Balaguer et al., 2001; Paula and Pausas, 2006). Under undisturbed conditions, it can reach several meters of height. However, due to its excellent drought resistance and resprouting ability, it frequently grows as a pioneer shrub in extended dense clumps of 1 to 1.5 m height in highly disturbed, fire prone, and dry scrublands. In the present study, we used eighty 5-year-old QC saplings originating from 4 scrubland populations along a north/east–south/west transect in the French Mediterranean region (Figure 1). According the Köppen–Geiger climate classification system, all sites lie in the Mediterranean CSA climate zone characterized by hot and dry summers. However, data recorded from the nearest meteorological stations suggest differences in the local climate with site 2 (Gabriac (G)) having the less and site 4 (Port-La-Nouvelle (P)) having the most arid climates (Supplemental Table S1). In all collection sites, QC dominated the vegetation cover. The accompanying vegetation was mainly composed of Q. ilex trees, Buxus sempervirens (boxwood), and Juniperus oxycedrus (cade) shrubs, and in the understory, of Brachypodium retusum (Mediterranean false brome), Cistus albidus (grey-leaved rockrose), Asparagus acutifolius (wild asparagus), Smilax aspera (rough bindweed), Rosmarinus officinalis (rosemary), and Thymus vulgaris (thyme). Boxwood and false brome are isoprene emitters, while cade, rosemary, thyme, and rockrose are weak emitters of MTs and sesquiterpenes, respectively (Owen et al., 2001; Bracho-Nunez et al., 2013).

The acorns were collected in fall 2006 from individual sprouts of QC clumps keeping a minimum distance of 5 m per sample to ensure that no acorns were sampled from same individuals. Plants were grown from the collected acorns in 5 L plastic pots with a mixture of argilo-calcareous soil, sand, and peat moss compost (pH = 5.5–6.5). Potted plants were kept outside in the institute garden, where they were irrigated during summer and occasionally fertilized with Osmocote Plus 12–14 M (15% N, 3.5% P, 9% K, 1.2% MG plus trace elements). The experiments were run during the months of March to June 2012. Twenty saplings were randomly selected from each population, avoiding, however, individuals with visible signs of disease or reduced growth. One month before the experiments started, the plants were transferred to a greenhouse at an approximate day/night temperature of 25°C/15°C to initiate bud break.

The mature current-year leaves of 20 individual saplings per site were screened once for BVOC emissions. In addition, we made repeated measurements on same individuals in order to know the extent the emission rate and composition vary with time, leaf age, and leaf position: We measured BVOCs on 6 plants at the beginning and at the end of the experiment (after ca. 50 days) using different current-year leaves each time. Furthermore, on 4 plants we measured emissions from leaves of 3 different cohorts (current-year, 1-year, and 2-year-old).

BVOC emissions and CO₂/H₂O gas exchanges were measured on individual plants by enclosing 2–4 terminal, fully developed leaves of a twig in a flat dynamic, temperature, and light-controlled chamber (Vol. about 105 mL, see Staudt and Lhoutellier, 2011, for a detailed description) located in a laboratory close to the experimental garden of the institute. Temperature and incident light were maintained at standard conditions of 30°C and 1,000 μmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD) to directly measure the basal emission rates (BER; also called emission factor) as applied in emission inventories. The leaves were placed perpendicular to the light to ensure homogenous light distribution on the adaxial surface of the leaves. Compressed air (Ingersoll Rand compressor, model 49810187) was previously cleaned and dried by a clean air generator (Airmopure, Chromatotec, St Antoine, France), then rehumidified to about 30% relative humidity by directing an adjustable portion of the air stream through a bypass with a washing bottle. The inlet flow was maintained at a constant rate of 700 mL min⁻¹ by a Brooks Mass Flow Controller, resulting in a chamber air residence time of ca. 10s. All tubing was made of PFA with the sampling lines heated at a constant temperature of 45°C. The whole system was air-cleaned throughout the night and the absence of background peaks in the empty chamber was regularly checked. Leaves were illuminated with an LED lamp (LX60 Heliospectra AB, Göteburg, Sweden). PPFD was measured with a quantum sensor (LI-COR, PAR-SB 190, Lincoln, NE, USA) located next to the chamber. Leaf and chamber temperatures were monitored with 2 thermocouples of type E (Chrom-Constantan, OMEGA Engineering, Manchester, UK). All data were stored on a 21× datalogger (Campbell Scientific Ltd., Shepshed, UK). Leaf gas exchanges of CO₂ and water vapor were monitored using 2 coupled CO₂/H₂O infrared gas analyzers (Li7000 and Li640; LI-COR Inc., Lincoln, NE, USA). The analyzers were calibrated with a certified CO₂ calibration gas (549 ppm, Air Liquide, France) and a dew point generator LI-610 (LI-COR Inc., Lincoln, NE, USA). The CO₂/H₂O gas exchange parameters photosynthesis (net CO₂ assimilation), transpiration, stomatal conductance, leaf internal CO₂-concentration, and photosynthetic water use efficiency (WUE) were calculated according to von Caemmerer and Farquhar (1981).

VOCs were sampled and analyzed by 3 independent methods. Isoprene was measured online with a Chromatotec AirmoVOC C2-C6 Gas Chromatograph (Chromatotec, St Antoine, France) equipped with a Flame Ionization Detector (FID). The instrument continuously drew air from the chamber outlet via a 1/8 inch PTFE tubing at a flow of 12 mL min⁻¹. For isoprene measurement, the chamber air was directed for 5 min into the internal multi-phase VOC trap maintained at –10°C, which was subsequently flash-heated for 2 min to release isoprene into a fused silica PLOT Al₂O₃/KCl column. The temperature program of the oven was 1 min at 40°C, 15°C increase per min up to 180°C, then 20 min at 180°C. MTs and semi-volatiles were measured offline with a Chrompack CP9003 GC-FID equipped with a Chrompack TCT4002 thermo-desorber (all Varian Inc.). For VOC measurement, 1 L of chamber air was sampled (0.1 L min⁻¹ for 10 min) on a glass cartridge filled with 200 mg Tenax TA (20–35 mesh, Agilent, Geneva, Switzerland). The sampled VOCs were separated on a Chrompack Sil 8CB low bleed capillary column (30 m × 0.25 mm) using the following temperature program: 3 min at 40°C, 3°C min⁻¹ to 100°C, 2.7°C min⁻¹ to 140°C, 2.4°C min⁻¹ to 180°C, 6°C min⁻¹ to 250°C. Finally, MT enantiomers were measured offline by GC analyses coupled with mass spectrometry using a Shimadzu QP2010 Plus GC-MS equipped with Perkin-Elmer Turbomatrix thermodesorber. One liter of air (0.1 L min⁻¹ for 10 min) was sampled on Perkin Elmer stainless steel cartridges filled with 300 mg Tenax TA. Enantiomers were analyzed on an Agilent Cyclodex-B column (30 m × 0.25 mm, 0.25 µm). The temperature program was 5 min at 40°C, 1.5°C min⁻¹ to 100°C, 20°C min⁻¹ to 200°C. Carrier gas was helium in the GC offline analyses (1 mL min⁻¹) and hydrogen in the GC online analyses. The online and offline GC-FIDs were calibrated by means of a custom-made, temperature-controlled permeation device equipped with certified permeation tubes, one containing isoprene and one α-pinene (all Chromatotec Group, St Antoine, France). Carrier gas was high purity nitrogen. The primary standard gas was introduced in the empty plant chamber and further diluted with clean air to achieve different target concentrations. The calibration factor (peak area per ng) of each VOC was derived from the slope of multi-point calibrations (see Supplemental Figure S1 for examples). Since FID is a relatively unspecific detector, the calibration factor obtained for α-pinene was extrapolated to other MTs measured by the offline GC-FID. In addition to the gas phase calibration, liquid standards were used to calibrate the off-line GC-MS and GC-FID. Authentic standards of α-pinene, sabinene, β-pinene, myrcene, limonene, and eucalyptol (Fluka Chemie AG, Buchs, Switzerland; Roth, Karlsruhe, Germany; purity ≥ 99%, except myrcene [approximately 90%]) were stepwise dissolved in methanol to achieve realistic concentrations. For calibration, an aliquot of a freshly prepared standard was injected onto the glass wool plug of an adsorption cartridge and then purged with pure nitrogen at a flow rate of approximately 50 mL/min for 6 min to disperse the standard into the adsorption bed. Retention indices and mass spectra were used to identify individual VOCs and their enantiomers (see Supplemental Figure S2 for examples of chromatograms). To generate mass spectra and retention times specific to our methods, we introduced in the chamber system homemade permeation tubes containing pure standards of the main emitted VOCs.

After measurements, leaves of the measured shoots were harvested to determine fresh and dry weights. Dry weight was determined after drying for at least 48 h in a ventilated oven at 60°C. The projected leaf area was measured and calculated using a scanner and the software SigmaScan. These data were used to calculate the leaf dry mass per projected leaf area (LMA, g m⁻²) and relative leaf water content (RWC: [fresh weight—dry weight]/fresh weight × 100). Emission rates were expressed on a leaf area or dry weight basis and calculated according to the following equation:

where BER_VOC is the basal emission rate of a VOC, F is the chamber air flow rate, C_plant is the concentration of the VOC measured in the chamber with the enclosed leaves of a sapling, µC_empty is the mean concentration of the VOC obtained from empty chamber measurements, and LA and LDW are the projected leaf area and leaf dry weight of the enclosed leaves, respectively. The air in the empty chamber was sampled and analyzed at least once a week and occasionally several times a day during the periods of measurements. The results showed the presence of a few peaks in trace amounts coeluting with some of the emitted isoprenoids, whose concentrations were relatively stable throughout the day and the experimental period. The mean concentrations of these VOCs were used for subtraction. Emission rates of isoprene were calculated from the online GC-FID data and those of all other BVOCs from the offline GC-FID data. The GC-MS data were only used to determine the ratios of enantiomers and to support compound identifications. In the present study, we considered only the enantiomeric ratios of (+)- and (−)-α-pinene, and (+)- and (−)-β-pinene, which were completely separated and showed no overlap to other BVOCs emitted by QC. The enantiomeric ratios obtained from GC-MS analyses were converted to absolute emission rates by multiplying the enantiomeric fractions with the α- and β-pinene emission rates obtained from the GC-FID measurements. To check the comparability of the 2 analytical methods, we also quantified the total emission rates of pinenes from the GC-MS analyses calibrated with liquid standards. The results were in good agreement with the GC-FID data (α-pinene: R² = 0.98; β-pinene: R² = 0.92).

Statistical tests were made with Statistica software and XLSTAT Addinsoft. Intraspecific variability of the emission profiles was evaluated with hierarchical cluster analysis (dendrograms) using Ward’s technique with Euclidean distance measure. Scatter plots and linear correlations between relative VOC emissions (percentages) were made to assess which were cosynthesized in QC. χ² analysis of a contingency table was used to compare frequencies of the emission types (chemotypes) between the populations. One-way analysis of variance (ANOVA), or Kruskal–Wallis test when data did not meet the requirements for parametric tests, were performed to test whether measured variables significantly differed between populations and chemotypes. Student or Mann–Whitney rank sum tests were performed to compare replicate measurements of emission rate and composition made on the same plants. The differences between groups of measured variables were considered to be significant at the level α = 0.05.

Under standard conditions, the amount of VOCs released from the leaves of 80 QC individuals varied by more than an order of magnitude between 53 and 1,643 ng m⁻² s⁻¹ (Supplemental Figure S3; Supplemental Data File 1). The main emitted VOCs were the MTs myrcene, α-pinene, β-pinene, sabinene, eucalyptol, and limonene, which together averaged 744 ± 89 SE ng m⁻² s⁻¹ (5.47 ± 0.65 nmol m⁻² s⁻¹; 28.1 ± 3.2 µg g⁻¹ h⁻¹) and contributed 96% ± 2% of the total VOC release. The remainder consisted of the MT β-ocimene, the sesquiterpene germacrene D and the green leaf volatiles (Z)-3-hexenyl acetate and (Z)-3-hexenol. In addition, isoprene was emitted at low rates (12.9 ± 2.3 ng m⁻² s⁻¹). Both, (−)- and (+)-enantiomers of α- and β-pinene were observed in the emissions of QC with (+)-pinene enantiomers dominating over (−)-pinene enantiomers. Mean emission rates of the pinene (+)-enantiomers were significantly higher than their respective (−)-enantiomers (Student tests, P < 0.01; n = 80). Among the 80 individuals, 59 (74%) emitted more (+)- than (−)-α-pinene, and 57 (71%) more (+)- than (−)-β-pinene. Except for a few cases, the enantiomers of α- and β-pinene were correlated; leaves emitting predominantly the (+)-enantiomer of α-pinene, emitted also predominantly the (+)-enantiomer of β-pinene (R² = 0.77, n = 80) and vice versa for the (−)-enantiomers (R² = 0.61, n = 80). The few exceptions to this rule were mainly observed when pinene emissions were low and hence more prone to imprecisions.

Mean net-photosynthesis, stomatal conductance, and WUE (±SE) were respectively 8.7 ± 0.8 μmol m⁻² s⁻¹, 111 ± 12 mmol m⁻² s⁻¹, and 2.98 ± 0.12 mmol mol⁻¹. Mean LMA was 95 ± 4 g m⁻² and RWC 51% ± 1%. MT and isoprene emission rates were slightly correlated with photosynthesis rates (r² = 0.13 and 0.14; P = 0.001; Supplemental Figure S4). Assuming that the carbon of all volatile isoprenoids stems from ongoing photosynthesis, about 0.9% ± 0.1% of net-assimilated carbon was lost by their emission.

Repeated measurements made on the same or different leaf cohorts revealed that within plant emissions varied mostly quantitatively and much less so qualitatively. Total emission rates differed sometimes by more than 100% between leaf cohorts (Supplemental Figure S5a) and season (sampling period; Supplemental Figure S5b). However, there was no consistent trend in these emission changes (Student tests; P > 0.05). Despite large differences in absolute emission rates, the relative emission composition of the major MTs varied little between within-tree replicates. This, together with the huge compositional differences observed between trees, suggests that the capacity of the trees to produce these major terpenes is under genetic control. Therefore, we excluded trace compounds and isoprene from further data analysis and focused on the major MTs to detect the presence of chemotypes in QC populations.

Hierarchical cluster analysis yielded 4 principal groups (chemotypes) according to the MT emission profile (Figure 2). The most frequent type (51 trees = 64%) hereafter referred to as Pinene type included the most diversified emission profile in terms of the percentage contributions of single MTs. Major compounds were α-pinene (31.3% ± 5.7% SD) and β-pinene (25.7% ± 3.4%) followed by myrcene (17.7% ± 5.6%), sabinene (12.7% ± 4.7%), and eucalyptol (9.1% ± 5.8%). Limonene contributed only a minor fraction in this chemotype (3.4% ± 1.1%). The second frequent cluster named Myrcene type (20 trees = 25%) was characterized by high proportions of myrcene (78.2% ± 16.4%) with minor though variable proportions of all other MTs (α- and β-pinene: 6.9% ± 6.7% and 7.3% ± 5.5%, sabinene: 3.0% ± 3.3%, limonene: 3.0% ± 1.7%, and eucalyptol: 1.6% ± 2.1%). The 2 remaining clusters were less abundant: the relatively homogenous Eucalyptol-type (6 trees = 7.5%) characterized by high proportions of eucalyptol (41.3% ± 4.6%) and sabinene (22.8% ± 2.5%), and a rare Limonene type (3 trees = 3.8%) containing large amounts of limonene (65.1% ± 15.9%). Cluster analysis without differentiating pinene enantiomers resulted exactly in the same major chemotypes (Supplemental Figure S6). However, the inclusion of pinene chirality clearly strengthened the distinction of the major chemotypes and revealed several subtypes within main clusters. Thus, within the Pinene type a small subcluster of plants could be identified, which emitted relative high amounts of (−)-pinenes and sabinene ((−)-Pinene/Sabinene subtype), while the profiles of all other pinene type emitters predominantly released (+)-pinene enantiomers ((+)-Pinene subtype). Further, within the Myrcene type, 2 subtypes could be distinguished: Half of the individuals emitted almost exclusively myrcene with only traces of pinenes predominantly in their (−)-form (Myrcene subtype), whereas the emissions of the other half included higher fractions of pinenes mostly in their (+)-form (Myrcene/(+)-Pinene subtype). A similar pattern can be seen within the rare Limonene type. The sole individual emitting predominantly limonene contains mainly (−)-pinenes as minor constituents, while in the 2 individuals showing a more mixed profile contained predominantly (+)-pinenes. Finally, the Eucalyptol type is distinct from the pinene type not only by its high proportions of eucalyptol and sabinene, but also by the predominance of (−)-pinenes in its profile. Correlation analyses corroborated the results of the cluster analysis. For example, a plot of relative sabinene emissions against those of α-pinene enatiomers shows that within the Pinene-type, (+)-Pinene-, and (−)-Pinene/Sabinene subtypes are distinguished by the positive correlation between (−)-α-pinene and sabinene proportions (Figure 3).

Apart from the emission composition, chemotypes were not significantly different for most of the measured variables. Thus, there was no relation between the emission type and the BER, photosynthesis, stomatal conductance, LMA, or RWC. The sole significant difference was observed in the WUE (ANOVA, P = 0.033) with the Myrcene-type having the highest (3.24 ± 0.59 mmol mol⁻¹) and the Eucalyptol-type the lowest values (2.74 ± 0.36 mmol mol⁻¹).

χ² analysis of the contingency table suggests an uneven geographical repartition in the frequency of the main chemotypes (Table 1). The B population was almost exclusively composed of Pinene chemotypes, of which the great majority (18 of 19) belonged to the (+)-Pinene subtype. By contrast, Myrcene and Pinene types occurred in about similar frequencies in the P and G populations. In addition, the P population contained 2 specimens of the very rare Limonene type absent in the G and B populations. All major chemotypes were present in the S population with a predominance of the Pinene type and a relative strong presence of the second rarest Eucalyptol type. Concerning the frequency of the subtypes, the P population held a high fraction of the Myrcene subtype together with a relative high fraction of the (−)-Pinene/Sabinene subtype, whereas in the G and S populations the Myrcene/(+)-Pinene and the (+)-Pinene subtypes were more frequent. Kruskal–Wallis tests performed with the relative emission rates (proportions) confirmed the differences in the mean terpene composition released by the 4 populations (Table 2). As expected, the average profile emitted by the B population contained significantly higher proportions of pinenes than the other populations, in particular the P population. Regarding pinene chirality, there were significant population differences in both the relative proportions of individual enantiomers and the enantiomeric ratios, with again the most contrasting profiles between the B and P population.

The 4 populations also differed in several other measured variables including the BER of the sum of major MTs, total VOC and isoprene; further the photosynthesis and stomatal conductance rates, carbon-losses and RWCs (Table 3). However, contrary to the emission composition, significant differences of these variables were all related to the S population, whose leaves exhibited an average lower physiological activity than the leaves of most other populations. Besides, the leaves of the saplings from the S and the mesic G populations had the highest RWCs.

BVOC emissions from QC have already been investigated in previous studies, though mostly on a few individuals of the same population (Llusià and Peñuelas, 2000; Ormeño et al., 2007; Ormeño et al., 2009; Olivier et al., 2011; Staudt and Lhoutellier, 2011; Bracho-Nunez et al., 2013; Llusià et al., 2013). All report MTs being the major VOCs emitted by this species in agreement with our results. The emission factors observed in our study are in the upper range of those reported previously for this species. Minor emissions of sesquiterpenes were also frequently reported before. Only a sole study reported minor emissions of isoprene (Bracho-Nunez et al., 2013), possibly because its measurement was not included in the other studies. The low emissions of isoprene observed in our study (ca. 2% of total emissions) and that of Bracho-Nunez et al. (2013) might stem from its non-enzymatic formation by divalent metal cations (Oku et al., 2022).

In terms of the emitted MT composition, all previous studies reported pinene, myrcene, limonene, and sabinene as the most abundant compounds. However, their relative contributions differed significantly between some studies (e.g., Olivier et al., 2011; Llusià et al., 2013). Our results show that this apparent inconsistency in the reported emission composition can be due to the inherent chemical polymorphism of MT synthesis in Kermes oak. The strong and discontinuous variation of the emission pattern of 80 individuals grown and measured under equal conditions suggest the existence of 4 major chemotypes with several subtypes in French QC populations. The frequency of chemotypes as well as the mean emission composition significantly differed among the 4 investigated QC populations (Tables 1 and 2) without apparent geographical or climatic clines. All 4 major chemotypes and most subtypes were present in the 2 most distant populations, S and P, while the intermediate B population was composed almost entirely of the (+)-Pinene subtype. This homogenous chemical profile of the B population may be due to limitations in genetic exchanges associated with the size and fragmentation of QC populations in that site. In addition, the regional wind system and local topography could play a role, because oaks are wind pollinated and the nuclear encoded genes of MT synthases (Memari et al., 2013) are maternally and paternally inherited. The most southwestern P and most northeastern S populations are located in the corridors of strong land winds called “Tramontane” and “Mistral” (Obermann et al., 2018), which may favor long distance transport of pollen and hence inter-population genetic exchanges. These north winds are much less pronounced in the 2 intermediate populations B and G, which are sheltered by mountains and are also much closer to the northern limit of the species’ range than the populations S and P (Figure 1).

Chemical polymorphism has already been observed in other MT emitting oak species, namely Q. ilex, Q. suber, and Q. afares (Staudt et al., 2001; Staudt et al., 2004; Loreto et al., 2009; Welter et al., 2012; Staudt et al., 2022). Compared to these, QC populations are distinguished by a high presence of Eucalyptol and Myrcene chemotypes and a low presence of the Limonene type (Table 4).

Individuals emitting eucalyptol in high fractions were never observed in the other oak species and therefore seem to be specific to QC. Myrcene emitters were found only in Q. ilex populations suggesting that this species is more closely related with QC. Indeed, studies using genetic markers classified QC and Q. ilex in a common section called Ilex, different to that of Q. suber and Q. afares belonging to the section Cerris (e.g., Hipp et al., 2020). However, genetic markers also revealed a large intraspecific variability among provenances sometimes blurring the discrimination of single oak species including QC (Piredda et al., 2011). Q. ilex and QC frequently cohabit on calcareous substrates and hybridization between the 2 species can occur, albeit rarely (Ortego and Bonal, 2010). Interestingly, in our previous study on Q. ilex (Staudt et al., 2001), the population with the by far highest proportion of myrcene emitters was found at the same site as in the present study on QC (i.e., Port-La-Nouvelle), possibly reflecting some gene flow between these 2 species.

Chemotypes may also designate ecotypes. Here, the QC chemotypes differed only in their WUE with the Myrcene type having the best efficiency to fix CO₂. Its high frequency in the warmest and most arid P site suggests that this type may represent an adaptation to local growth conditions. Whether the high fraction of myrcene in its emission is only a marker of an ecotype or is directly involved in WUE determining processes remains to establish. In analogy to research on isoprene emissions, the chloroplastic MT production in oak leaves might play a role in abiotic stress signaling, growth/defense regulation (Monson et al., 2021; Dani et al., 2022), and stomatal control (Lantz et al., 2019), whose effectiveness may differ among singular BVOCs (Copolovici et al., 2005). Among the 6 major MTs emitted by QC, myrcene is the only acyclic and most reactive one (Atkinson and Arey, 2003), and eucalyptol is the less reactive and most water-soluble compound (Sander, 2015). However, the Myrcene type was also present in the other populations and in particular, in the most mesic and coldest G site, where QC propagation is more likely limited by freezing events and competition with more shade and cold tolerant tree species. Furthermore, neither WUE nor the chemotypes were related to the foliar BER, as for example observed for isoprene emissions from poplar genotypes by Guidolotti et al. (2011). Instead, mean BER was significantly lower in the most northeastern S population independent of its chemotype composition. Possibly the low emissions of several specimen of this population was due to a delay in their leaf maturation, since they expressed a general lower physiological activity at the time of measurements and repeated measurements on the same plants suggest that the foliar BERs could have varied considerably during the experimental period.

The differentiation of pinene enantiomers clearly helped us to characterize the chemical polymorphism in QC BVOC emissions. The coordinated occurrence of α- and β-pinene enantiomers suggests that MT synthases are more specific for chiral than for structural isomers, as already described in various previous studies (e.g., Phillips et al., 2003; Martin et al., 2004; Krause et al., 2013). Assuming that several MT synthases can be active in QC individuals, the observed emission variations can be explained by a combination of 5 putative enzymes, which respectively produce predominantly myrcene, limonene, eucalyptol, (+)-pinenes, and sabinene/(−)-pinenes. Here, as well as in the aforementioned studies on other oak species, individuals showing emissions strongly dominated by a sole compound were found only for myrcene and limonene but never for the other MTs (Figure 2; Staudt et al., 2001; Staudt et al., 2004; Welter et al., 2012). This indicates that highly product-specific enzymes for myrcene and limonene in oaks exist, while the other MTs and in particular pinenes are likely synthesized by multiproduct enzymes. Indeed, in a study on MT-synthase activities in Q. ilex, Fischbach et al. (2000) observed that the biochemical characteristics of the limonene-forming enzyme differed from that of forming pinenes plus sabinene. Furthermore, in a follow-up study Fischbach et al. (2001) isolated a gene encoding a single-product myrcene synthase from Q. ilex leaves.

Our study revealed a marked intraspecific variability at plant and population level in the rates and chemical composition of volatiles emitted by QC saplings grown and assayed under the same conditions. Knowing this variability will help to improve regional emission inventories in the Mediterranean region, especially to assess the uncertainty when assigning compound-specific BERs to BVOC emitters (e.g., see Kemper Pacheco et al., 2014). Given that the mean MT emissions from populations significantly differed in their composition of structural and chiral isomers, the chemical fingerprint of the air over QC populations may vary depending on the prevalence of chemotypes. Whether this has any real impact on regional air chemistry has yet to be demonstrated. Nevertheless, our results provide an additional explanation for spatial variations in the enantiomeric ratios of VOCs in the air, as observed, for example, by Williams et al. (2007). Specifically, they imply that such variation is not necessarily a marker of different types of emission sources or related to stress (e.g., Byron et al., 2022). Resulting simply from the intraspecific polymorphism of constitutive terpene production, it could actually blur other spatial changes in the enantiomeric ratios that are related to variation in source types.

The chemical polymorphism in QC is similar, though somewhat more pronounced than those reported for other MT producing oaks. We attribute this variability to inherent differences in the level of active MT synthases within oak populations, reflecting their degree of relatedness and genetic diversity. Our results are insufficient to deduce an ecological significance of this chemical polymorphism in oaks. Theoretically, the chemical diversity of oak emissions could be important to insects and vertebrates that use olfactory cues in addition to visual cues to find their specific food, mates, or habitat (e.g., Rubene et al., 2022). Changes in the olfactory environment due to constitutive emissions from vegetation cover could affect their behavior, even if the plant emissions do not contain volatiles released by the animal’s target, such as herbivore-induced VOCs or pheromones (Conchou et al., 2019 and references therein). Further studies implying stress conditions are needed to detect potential associations between chemotypes and ecotypes (e.g., see Huang et al., 2010). On the other hand, it is well documented that the product pattern of terpene synthases in plants can easily change by gene duplication and a few mutations in the sequence encoding the active center of the enzymes (e.g., Christianson, 2017; Xu et al., 2021). Thus, we believe that the ubiquitous chemodiversity in the constitutive MT emission in oak populations does not necessarily result from selection but rather witnesses an ongoing, ecologically neutral diversification of a rapidly evolving class of metabolites (Firn and Jones, 2003; Owen and Penuelas, 2005). However, nonemitting individuals, which have been rarely observed in Q. afares and Q. suber populations were not found in the present study. This points to a strong negative selection of mutants deficient in constitutive MT production, perhaps due their role in maintaining a balance in the biosynthesis of isoprenoid precursors and their partitioning to other isoprenoid classes (Krause et al., 2023).

The data of this study are in the Supplemental Data File 1 of the article.

The supplemental files for this article can be found as follows: Supplemental material is included in 2 files: (1) Supplemental Material 1.DOC contains Supplemental Table S1 and Supplemental Figures S1–S6; and (2) Supplemental_Data File 1.XLS contains the data used in the study.

The authors are grateful to Coralie Rivet and Bruno Buatois for their valuable help with the GC-MS analyses.

This research was supported by the Agence Nationale de la Recherche grant number ANR-10-LABX-04-01.

The authors have declared that no competing interests exist.

Designed, planned, and conducted the experiments, performed the data analysis, and wrote the manuscript: MS, IV. Both authors approved the submitted version for publication.

How to cite this article: Staudt, M, Visnadi, I. 2023. High chemodiversity in the structural and enantiomeric composition of volatiles emitted by Kermes oak populations in Southern France. Elementa: Science of the Anthropocene 11(1). DOI: https://doi.org/10.1525/elementa.2023.00043

Domain Editor-in-Chief: Detlev Helmig, Boulder AIR LLC, Boulder, CO, USA

Associate Editor: Alex Guenther, Department of Earth System Science, University of California Irvine, Irvine, CA, USA

Knowledge Domain: Atmospheric Science