Foliar Mono- and Sesquiterpene Contents in Relation to Leaf Economic Spectrum in Native and Alien Species in Oahu (Hawai i)

Transcription

1 J Chem Ecol (2010) 36: DOI /s z Foliar Mono- and Sesquiterpene Contents in Relation to Leaf Economic Spectrum in Native and Alien Species in Oahu (Hawai i) Jordi Sardans & Joan Llusià & Ülo Niinemets & Sue Owen & Josep Peñuelas Received: 18 November 2009 / Revised: 03 January 2010 / Accepted: 5 January 2010 / Published online: 11 February 2010 # Springer Science+Business Media, LLC 2010 Abstract Capacity for terpene production may confer advantage in protection against abiotic stresses such as heat and drought, and also against herbivore and pathogen attack. Plant invasive success has been intense in the Hawaiian islands, but little is known about terpene content in native and alien plant species on these islands. We conducted a screening of leaf terpene concentrations in 35 native and 38 alien dominant plant species on Oahu island. Ten (29%) of the 35 native species and 15 (39%) of the 38 alien species contained terpenes in the leaves. This is the first report of terpene content for the ten native species, and for 10 of the 15 alien species. A total of 156 different terpenes (54 monoterpenes and 102 sesquiterpenes) were detected. Terpene content had no phylogenetic significance among the studied species. Alien species contained significantly more terpenes in leaves (average ± SE=1965±367 μgg 1 )thannativespecies (830±227 μgg 1 ). Alien species showed significantly higher photosynthetic capacity, N content, and lower Leaf Mass Area (LMA) than native species, and showed higher total terpene leaf content per N and P leaf content. Alien species, thus, did not follow the expected pattern of excess carbon J. Sardans (*) : J. Llusià : J. Peñuelas Global Ecology Unit CSIC-CEAB-CREAF, Facultat de Ciencies, Edifici C, Universitat Autònoma de Barcelona, Bellaterra, Spain j.sardans@creaf.uab.cat S. Owen Centre for Ecology and Hydrology Edinburgh, Bush Estate, Penicuik, EH26 0QB Scotland, Great Britain Ü. Niinemets Estonian University of Life Sciences, Institute of Agricultural and Environmental Sciences, Kreutzwaldi 1, Tartu, Estonia in comparison with native species. Instead, patterns were consistent with the nutrient driven synthesis hypothesis. Comparing alien and native species, the results also support the modified Evolution of Increased Competitive Ability (EICA) hypothesis that suggests that alien success may be favored by a defense system based on an increase in concentrations of less costly defenses (terpenes) against generalist herbivores. Keywords Hawaiian Islands. Terpene content. Nitrogen. Phosphorus. Alien species. Native species. LMA. Photosynthetic capacity. Monoterpenes. Sesquiterpenes. Nutrient driven hypothesis. Excess carbon hypothesis. Modified EICA hypothesis Introduction Plant invasion is an important component of current global change (Mooney and Hobbs 2000). Chemical factors such as terpenes can be involved in the competition between alien and native plant species. For example, Barney et al. (2005) stated that the terpene production capacity of Artemisia vulgaris can be a key factor in its establishment and proliferation in introduced habitats by phytotoxic effects on native species. Many studies have investigated the physiological and ecological significance of terpenes in plants. Protection, defense, and infochemical function have been highlighted as roles of terpenes (Llusià and Peñuelas 2001; Wheeler et al. 2002; Peñuelas and Llusià 2003, 2004). Examples of these roles are photoprotection (Peñuelas and Munné-Bosch 2005), thermotolerance (Sharkey and Singsaas 1995; Peñuelas and Llusià 2001, 2002; Copolovici et al. 2005; Peñuelas et al. 2005), protection against drought stress

2 J Chem Ecol (2010) 36: (Llusià and Peñuelas 1998; Kainulainen et al. 1991), and non-specific antioxidative capacity, whereby terpenes protect photosynthetic membranes against peroxidation and reactive oxygen species such as singlet oxygen (Loreto and Velikova 2001; Peñuelas and Llusià 2002; Loreto et al. 2004; Munné-Bosch et al. 2004; Llusià et al. 2005). Although relative performance often depends on growth conditions, invaders are more likely to have higher leaf area and lower tissue construction costs that increase productivity, and also greater phenotypic plasticity that is advantageous in disturbed environments (Daehler 2005). Foliar traits such as higher photosynthetic capacity per dry mass (A mass ) and lower leaf construction costs associated with a lower leaf mass per area (LMA) partly explain the success of alien plant species (Baruch and Goldstein 1999; Funk and Vitousek 2007), since they may contribute to faster growth rates for invaders and confer a competitive advantage over native species (Reich et al. 1997; Peñuelas et al. 2010). Similarly, invasive plant species in Hawai i have been found to have higher foliar N and P concentrations than native species (Peñuelas et al. 2010). Changes in nutrient availability can affect terpene production (Son et al. 1998; Kainulainen et al. 2000; Lee et al. 2005). Greater terpene production in plants with higher nutrient concentration and photosynthetic rates can be expected from the nutrient-driven synthesis hypothesis that predicts a larger enzyme production with greater cellular N and P availability. Higher nutrient availability usually is expected to translate into higher carbon fixation and activity of the enzymes involved in isoprenoid production (Harley et al. 1994; Litvak et al. 1996). In contrast, a lower production of terpenes as carbon based secondary compounds under higher nutrient availabilities can be expected from the carbon based secondary compounds (CBSC) hypthesis and the source-sink carbon-nutrient balance or excess carbon (CNB) hypothesis (Loomis 1932;Bryantetal.1983;HermsandMattson1992;Peñuelas and Estiarte 1998). These hypotheses assert that plants allocate carbon to secondary metabolism only after growth requirements are met, and that growth is constrained more by nutrients than by photosynthesis. According to these theories, the excess carbohydrates that accumulate in nutrient-limited plants when photosynthesis outpaces growth are diverted to the production of carbon-based secondary compounds (e.g., terpenes and phenolics). Phenotypic plasticity has been an important mechanism that enables alien plants to colonize exotic habitats, and recent studies indicate that alien plants also can evolve quickly (Maron et al. 2004). Some invasive trees and herbs have proved to be able to evolve in periods from 1 to 3 hundred years or less, reaching a faster growth capacity, and changing their chemical defense strategies (Rogers and Siemann 2004; Siemann et al. 2006). The main cause of this increase in fitness has been proposed as the Evolution of Increased Competitive Ability (EICA) hypothesis (Blossey and Nötzold 1995). It predicts that introduced species, which lose contact with their natural specialist herbivores, may evolve, thus decreasing their investment in anti-herbivore chemical defenses. This way, resources no longer needed for defense can be reallocated to other functions that provide a selective advantage in the novel habitat. Recent modifications in the development of increased competitive ability (EICA) hypothesis propose that since invasive genotypes still may experience attack by local generalist herbivores (Müller-Schärer et al. 2004), selection may favor a reduction in the expression of metabolically expensive chemical defenses effective against specialist herbivores, and increase the concentrations of less costly qualitative defenses, such as terpenes, that may be more toxic to generalist herbivores (Joshi and Vrieling 2005; Stastny et al. 2005). In this context, Johnson et al. (2007) have observed that when North American native populations of Solidago gigantea grow under the same environment conditions as alien European population of the same species, the native plants have lower monoterpene and diterpene contents than invasive plants. This suggests that terpene content might be related to alien success. The Hawaiian archipelago is the most isolated terrestrial region on Earth (Vitousek and Walker 1989), and is especially vulnerable to invasions by non indigenous species (Harrington and Ewel 1997). Alien plants in Hawai i have strong impact on native Hawaiian ecosystems and their highly endemic flora (Mack and D Antonio 2003; Hughes and Uowolo 2006). In these Islands, around 861 flowering plant species (47% of total Hawaiian angiosperm flora) are naturalized alien species (Wagner et al. 1999). As a result, approximately 25% of the Hawaiian native flora, 90% of which is endemic, has been listed as threatened or endangered. In fact, all tropical island ecosystems appear to be especially vulnerable to invasive species, and some experiments suggest that the high resource availability and the poor ability of native species to capture these resources, contributes to the vulnerability of island communities to the establishment and spread of alien species (Allison and Vitousek 2004). There are published reports of terpene contents in species that are aliens in Hawai i, but these studies have been conducted in other parts of the world (Ogunkoya et al. 1972; Schapoval et al. 1998; Kikuzaki et al. 2000; Wheeler et al. 2002; Chiang and Kuo 2002; Pino et al. 2005; Randrianalijaona, et al. 2005; Fernández and Torres 2006; Pachanawan et al. 2008). Generally, apart from scarce reports (Komai and Tang 1989) little is known about terpene content in Hawaiian native and alien flora. In this study, we conducted a screening of leaf terpene content in 35 native and 38 alien dominant Hawaiian plant

3 212 J Chem Ecol (2010) 36: species. Our aims were to: (i) estimate terpene content and composition of native and alien species that are dominant in Oahu, (ii) compare the mono- and sesquiterpene content of the 35 native plants with the content of 38 alien plants, (iii) compare the relationships of terpene content with the leaf traits defining leaf economics spectrum (Wright et al. 2004), such as photosynthetic rates (A mass ), leaf mass area (LMA), and C, N, P, and K leaf concentrations among native and alien species, and (iv) test the nutrient driven synthesis, excess carbon, and modified EICA hypotheses for terpene content in native and alien species. Methods and Materials Field Sites The study was conducted in May 2007 on Oahu, the third largest of the Hawaiian Islands. As typical of larger Hawaiian Islands, the climate is characterized by steep rainfall gradients over short distances (Müller-Dombois and Fosberg 1998). Lowlands at the leeward side have a pronounced dry summer season, while precipitation is distributed almost uniformly in lowland and mountain rain forests. Due to the oceanic tropical climate, interannual temperature oscillations are small with winters having on average 2 3 C cooler temperatures than summers. As large differences in the composition of native and alien vegetation occur in response to rainfall gradients, four sites with distinct precipitation regimes were selected for plant sampling in the leeward lowlands of Oahu, and at the leeward side of Koolau mountains (Table 1 and see detailed decription of the sampling sites, their climate and the studied species in Peñuelas et al. 2010). The four key soil types found across the sites rank according to the state of soil weathering as oxisols>ultisols> mollisols>inceptisols (Uehara and Ikawa 2000; Deenik and McClellan 2007). Mollisols exhibit the highest fertility, while more leached oxisols and ultisols with lower ph are among the soils with lowest fertility (Uehara and Ikawa 2000; Deenik and McClellan 2007). Inceptisols, the youngest soils, typically show weak profile development, and, depending on genesis, exhibit tremendous variability in fertility (Deenik and McClellan 2007). The Tantalus series inceptisols are of moderate to high fertility, while the inceptisols in rocky soils and mountainous land are of low fertility. Thus, in our study, the broad soil classes rank according to fertility as mollisols > inceptisols (Tantalus) > oxisols ultisols > inceptisols (mountainous soils). Study Species Altogether, 73 dominant,(35 native, and 38 alien) species were studied at four sites (Peñuelas et al. 2010). All native species sampled were evergreen, but in dry sites (St. Louis Heights, Hahaione Valley), four alien species were drought-deciduous (Desmodium incanum, Falcataria moluccana, Senna surattensis, Tabebuia rosea), and two were semi-deciduous (Haematoxylum campechianum, Leucaena leucocephala). The deciduous and semideciduous aliens were legumes, except for Tabebuia rosea (Bignoniaceae). Of the 73 studied species, 36 were trees, 29 shrubs, 3 woody vines to shrubs, 3 woody vines, one herb to subshrub, and one parastitic mistletoe (Korthalsella complanata). The distribution of species among the key life form classes was similar among native and alien species (14 alien and 15 native shrubs, 21 alien and 15 native trees; Peñuelas et al. 2010). Each species was sampled in triplicate, with twigs or small branches taken from 3 individuals for each species. Samples were cut with a sharp knife and cut again immediately with the excised stem under water, to prevent ingress of air to the xylem vessels and subsequent stress. In the lab, the excised stems were enclosed loosely in plastic bags to prevent water loss by transpiration prior to terpene extraction. Leaf extractions were conducted during the next 12 h. Species coordinates and sampling altitude were noted in each site by using GPS, and this information was used to link species locations to specific soil types and to derive location-specific climatic data. Long-term average monthly and annual precipitation, precipitation of the three driest months and annual precipitation, and average, maximum Table 1 Description of the study sites Site Coordinates Average ± SD a Average ± SD precipitation (mm) Average ± SD annual temperature ( C) altitude (m) Annual Three driest months Minimum Maximum n N A St. Louis Heights N, W 171± ± ± ± ± Hahaione Valley N, W 390± ±22 157±7 17.1± ± Tantalus 21 N, W 441± ± ± ± ± Wiliwilinui N, W 660± ± ± ± ± N number of species, N native species, A alien species a averages are based on the number of species sampled and species-specific locations. In statistical analyses, exact species-specific environmental data were used

4 J Chem Ecol (2010) 36: and minimum temperatures were estimated from high resolution climatic grids by using the database developed and continuously updated by Giambelluca and associates (Giambelluca et al. 1986; Cao et al. 2007). ARCGIS 9.1 was used to extrapolate between the isohyets (10 m square cells in the grid with appropriate elevation model), as applied previously in Hawaiian ecosystems (Porder et al. 2005; Dunbar-Co et al. 2009). Species were classified according to site preference as dry, dry-mesic, mesic, dry-wet, mesic-wet, and wet forest species. Species invasiveness was scored by using a fourlevel scale as 0 (native species), 1 (low invasiveness), 2 (moderate-high), and 3 (very high). These simplified scores were based on the Australia/New Zealand weed risk assessment (WRA) system (Pheloung et al. 1999) modified to Hawai i and other Pacific Islands (Daehler et al. 2004). For Hawaiian Islands, these scores are reported in Pacific Island Ecosystems at the Risk (PIER) project online database, maintained by U.S. Forest Service s Institute of Pacific Islands Forestry ( and on recent updates on species invasive potential in Oahu (Daehler and Baker 2006). The weed risk assessment is based on up to 49 questions about species biology. For 9 species that have not been scored in these assessments, weed risk assessment scores were derived based on the risk questionnaire ( As the risk assessment provides information of possible species invasiveness, but not on whether the species actually becomes invasive in the specific new habitat, finally, a simplified 3-level scale (1 3) was used to group aliens with varying invasive potential and known invasiveness throughout Oahu. (Daehler et al. 2004; Daehler and Baker 2006). Leaf Terpene Extraction and Analysis Leaf samples were crushed in liquid nitrogen with a Teflon pestle in a Teflon tube until a homogeneous powder was obtained. Between 2 4 ml of pentane (depending on the leaf type) were added before the pulp defrosted. The Teflon tubes were maintained airtight at 25 C during 24 h for full extraction. After this, a sample of each extract was put into a 300 μl glass vial. Samples were injected automatically into the GC-MS following a split of 0.5:80, thus allowing only 0.625% of the injected sample to enter the column. The column was an HP-5 crosslinked 5% PH Me Silicone (Supelco Inc.). Solvent delay was 3 min. The initial temperature of 40 C was increased immediately with a ramp of 30 C min 1 to 60 C. The second ramp was 10 C min 1 to 150 C, which was maintained for 3 min. The third ramp was 70 C min 1 to 250 C, which was maintained for 5 min. Carrier gas was helium at 0.7 ml min 1. The mass detector was used with an electron impact of 70 ev. Identification of monoterpenes was conducted by GC-MS and comparison with authentic standards from Fluka (Buchs, Switzerland), literature spectra, and GCD Chemstation G1074A HP. Calibration with common terpenes pinene, 3-carene, β-pinene, β- myrcene, p-cymene, limonene, sabinene (monoterpene), and humulene (sesquiterpene) standards was carried out once every 5 analyses. Standards were purchased from Sigma Aldrich (Gilingham, Dorset, UK). Terpene calibration curves (N=4 different terpene concentrations) were always highly significant (r 2 >0.99 for the relationships between signal and terpene concentrations). The most abundant terpenes had similar sensitivity (differences were less than 5%). Quantification of the peaks was conducted according to the amount of ion 93 in the compound and by using the calibration of the most similar mono- or sesquiterpene standard depending on the compound investigated. The total GC run time was 23 min. All sampling and analytical procedures were applied in the same way for native and alien species. Statistical Analyses The program Phylomatic (Webb and Donoghue 2005) was used to build a phylogenetic tree of the species studied (Fig. 1) as explained in Peñuelas et al. (2010). The stadistical significance of the genetic differences among different species in explaining the variability of the studied variables was calculated by employing Matlab with the PHYSIG module developed by Blomberg et al. (2003). Altitude was significantly correlated with all the main climate variables of each respective site (total annual precipitation, the precipitation of the three driest months, mean annual temperature, annual mean of the daily minimum temperatures, annual mean of the daily maximum temperatures, annual mean of monthly difference between the maximum and minimum temperature and annual mean of the coldest monthly temperature) (data not shown). To analyze the effects of all studied characteristics on foliage terpene contents, we conducted a general linear model (GLM) with site (4 different sample sites), species origin (native and alien), and soil type (5 different soil types) as independent categorical variables, altitude as independent continuous variable, and in the case of variables with phylogenetic fingerprinting, phylogenetic distances also were included as continuous independent factors. We introduced the factor site in the GLM design to extract the variability due to site. Since the origin of the studied species (native vs. alien) showed a significant phylogenetic signal (k=0.309, P=0.022) mainly due to the high abundance of alien species of the Orders Rosales, Lamiales, and Laurales, we conducted all the statistical analyses that had the origin of species as the independent factor both by an ordinary GLM without phylogenetic distance matrix and also with phylogenetic distance matrix even when the dependent variable had no significant phylogenetic signal. Thereafter, the model with a lower

5 214 J Chem Ecol (2010) 36: Fig. 1 Phylogenetic tree of the studied woody plant species obtained from PHYLOMATIC programme (Webb and Donoghue 2005). The scale depicts millions of years. The field sampling site is depicted between brackets to the right of each species. (SLH = St. Louis Heights. HV = Hahaione Valley. T = Tantalus. WR = Williwillinui Ridge). The origin (N = Native. A = Alien) is also depicted

6 J Chem Ecol (2010) 36: Akaike information criterion (AIC) was selected. To conduct these analyses we used Matlab with REGRESSIONV2 module (Lavin et al. 2008). We also conducted a PCA analysis with leaf economics spectrum variables (nutrients, A mass and LMA) and the different sets of species as cases. Then, we looked at the correlation between the factor scores that characterized the functional spectrum and total leaf terpene contents by using Statistica 6.0 software package (StatSoft, Inc. Tule, OK, USA). We employed the same rationale to analyze potential differences between native and alien species in terpene contents vis-à-vis leaf chemical, physiological, and anatomical traits. To analyze what variables are differently correlated between alien and native species, we conducted discriminant analysis among leaf total terpene contents and leaf traits by using Matlab with REGRESSIONV2 module (Lavin et al. 2008) and Statistica 6.0 software (StatSoft, Inc. Tule, OK, USA). To analyze the effects of leaf economics spectrum on terpene emisssion and whether there were different relationships between native and alien species, we also conducted a PCA analysis with the species as cases and leaf economics spectrum (nutrients, amass, and LMA) as variables and calculated the factor scores for each species. Thereafter, we conducted a correlation between the factor scores that characterized the functional spectrum and the total terpene contents. To compare possible differences in the proportion of species that produced and accumulated terpenes between the alien and native species set, we conducted a Chi-square test by using Statistica 6.0 software package (StatSoft, Inc. Tule, OK, USA). Results Foliage Terpene Concentration. Alien vs. Native Species Twenty-five (10 natives and 15 alien) of the 73 studied species contained at least one terpene (concentration above the detection limit of 0.6 ng g 1 in their leaves; Tables 2 and 3). No terpenes were detected in 48 of the 73 studied species (Appendix 1). Total terpene and total monoterpene concentrations were higher in the entire set of alien species than in the entire set of native species (P=0.02 and P=0.04, respectively) (Table 4, Fig. 2). In alien species, total terpene concentration was 1965±367 μg g 1 (4524±1225 μg g 1 when considering only storing species) and in native species it was 830±227 μg g 1 (2905±650 μg g 1 when considering only storing species). Total sesquiterpenes, cyclic monoterpenes, cyclic sesquiterpenes, and aromatic monoterpenes were also higher in alien than in native species but the differences were not significant (Table 4, Fig. 2). The greater average total terpene content in alien than in native species was due mainly to higher leaf monoterpenes in moderately-high invasive species and to higher sesquiterpenes in highly invasive species than in native species (Fig. 2). Neither the sampling site nor soil type had any significant effect on total terpene content (Table 4). The proportion of species that accumulated terpenes, although higher in alien species, was not significantly different between native (29%) and alien species (39%) (χ 2 =0.97, P=0.42). Several mono- and sesquiterpene compounds were found in 10 native species (Cheirodendron trigynum, Melicope clusiifolia, Melicope peduncularis, Metrosideros macropus, Metrosideros polymorpha, Metrosideros rugosa, Metrosideros tremuloides, Myrsine lessertiana, Myrsine sandwicensis, Syzygium sandwicensis), which had not been described previously as terpene accumulators to our knowledge. Syzygium sandwicensis accumulated 3 monoterpenes (camphene, E-β-ocimene, 3,7 dimethylocta-1,3,7-triene) and 11 sesquiterpenes (Tables 2 and 3) in its leaves. In the species of the genus Metrosideros, three monoterpenes (sabinene, 1,3,6-octatriene, 3,7- dimethyl E-β-ocimene) and 18 sesquiterpenes (Tables 2 and 3) were detected. In the two species of the genus Myrsine, 2 monoterpenes (limonene, myrcene) and 33 sesquiterpenes (Tables 2 and 3) were observed. In Cheirodendron trigynum, 11 monoterpenes and 11 sesquiterpenes were found (Tables 2 and 3). Finally, in the two Melicope species, 24 monoterpenes and 29 sesquiterpenes were found (Tables 2 and 3). Among the 15 alien species that accumulated terpenes in their leaves, three species Heliocarpus americanus (1 monoterpene, 7 sesquiterpenes, Tables 2 and 3), Schinus terebinthifolius (7 monoterpenes, 9 sesquiterpenes), and Persea americana (7 monoterpenes, 3 sesquiterpenes) had not been reported previously as terpene accumulators. Phylogenetic influence on the values of the variables was present only in 6 of the 156 different detected terpenes: β-cubebene, β-maaliene, epi-bicyclo-sesqui-phellandrene, γ-elemene, β-ocimene and l-β-pinene. Total terpene contents did not show a phylogenetic effect (data not shown). Relationships of Leaf Terpenes to A mass, LMA and Nutrient Leaf Concentrations and Climate No significant relationships of total terpenes with climatic characteristics and A mass were observed in the corresponding GLM analysis either in native or alien species (data not shown). Discriminant analysis of the total terpenes (TT) and leaf traits: LMA (Wilk s Lambda = 0.58 and P=0.002), leaf N concentration (Wilk s Lambda = 0.65 and P=0.008), leaf K concentration (Wilk s Lambda = 0.82 and P=0.1) and A mass (Wilk s Lambda = 0.67 and P=0.019), separated native and alien species (Fig. 3), showing that the set of native species had greater LMA and lower leaf economic traits and leaf terpenes than the set of alien species. Relationships of total

7 216 J Chem Ecol (2010) 36: terpene contents with the main leaf economic and structural traits did not differ between native and alien species (Fig. 3). There was no significant correlation between total leaf terpenes and the PC1 scores of each species obtained in the PCA analysis of leaf economic traits (LMA, Amass, N, K) within native plants (R=0.17, P=0.64) nor within alien plants (R=0.090, P=0.77) (Fig. 3). Discussion Terpenes in Hawaiian Plants This study provides novel information about terpene contents in Hawaiian native plant species. It also contributes to advance our understanding of terpene content in several alien species that have worldwide distributions (Sharma et al. 1999; Olajide et al. 1999; Ghisalberti 2000; Kikuzaki et al. 2000; Wheeler et al. 2002; Pino et al. 2005; Zhao et al. 2009). None of the 10 native species that accumulated terpenes has been reported previously as terpene-containing, at least to our best knowledge. At the genus level, only some species of the genus Syzygium had been reported as terpene accumulators (Chang et al. 1999). Among the 15 alien terpene-containing species, six had been previously identified as mono and sesquiterpene-containing species (Psidium guajava, Olajide et al. 1999; Lantana camara, Sharmaetal. 1999; Ghisalberti 2000; Pimenta dioica, Kikuzaki et al. 2000; Melaleuca quinquenervia, Wheeler et al. 2002; Mangifera indica, Pino et al. 2005; Ageratina adenophora, Zhao et al. 2009). In these cases, the terpenes reported previously and those found in the present research generally were the same, but with some differences. Some terpenes found in Lantana camara, suchas gurjunene or zingiberene, had not been reported previously in this species. Similarly, in Melaleuca quinquenervia, we found 7 terpenes (viridiflorol, 1 8 cineole, terpineol, pinene, β-pinene, γ-terpinene, terpinene) of the 10 already previously reported in this species (Wheeler et al. 2002), but in addition we also found 8 more monoterpenes and 22 sesquiterpenes (Tables 2 3). It is possible that the sampling technique may have induced some stress, which might have resulted in the production of these extra compounds. Five of the 17 alien species that accumulated several mono- and sesquiterpenes, Cinnamomum burmannii, Eucalyptus robusta, Psidium cattleianum, Rubus rosifolius, and Syzygium cumini (Tables 2 3) had not yet been reported as terpeneaccumulator species, although members of the same genus have been described as terpene accumulators (Chang et al. 1999; Olajide et al. 1999; Moore et al. 2004; Chaoetal. 2005; Yang et al. 2005; Malowicki et al. 2008). In another species, Pluchea carolinensis, known to have medical properties, terpene content has been suggested, but had not been described (Fernández and Torres 2006). In this study, 1 monoterpene and 3 sesquiterpenes were detected in this species. Finally, three alien species Heliocarpus americanus, Schinus terebinthifolius and Persea americana had not been reported previously as terpene accumulators. We did not detect a phylogenetic effect in the total terpene content among the set of studied species, nor in the content of the individual terpenes. Higher Terpene Content in Alien Species and No Relationship to Leaf Economics In regard to the number of species that accumulate terpenes, although the difference was not significant, the proportion of species that accumulated terpenes in leaves was slightly higher in alien (39%) than in native (29%) species. In regard to the absolute terpene leaf accumulation, alien species accumulated greater amounts in leaves, and also had greater N and P leaf concentrations than native species, and their ratio of terpene contents to the concentrations of these two elements were higher than in native species. Thus, the differences between alien and native species were proportionally greater in leaf terpene accumulation than in N and P leaf content. Collectively, these data suggest a greater investment in terpene production with respect to nutrient absorption and carbon fixation in alien compared with native species. Thus, alien species in Hawai i may have more productive leaves and invest more of their leaf primary production in terpene accumulation than native species. The discriminant analyses based on the relationships between total terpene accumulation and LMA and nutrient content significantly separated alien from native plants, thus reflecting that alien species have greater total terpene contents, N and K concentrations, and A mass and lower LMA than native species. When comparing native with alien species, a significantly higher leaf nutrient and terpene content are observed in alien species. This segregation suggests that these species may be using different resources and might also have greater capacities to capture and use nutrients. Despite this, however, there were no significant relationships between leaf terpene accumulation and leaf economic spectrum in either native or alien species. Nutrient Driving, Excess Carbon and EICA Related Hypotheses The comparison of alien and native species did not support the excess carbon hypotheses (Loomis 1932; Bryant et al. 1983; Herms and Mattson 1992; Peñuelas and Estiarte 1998). Alien species had higher N leaf contents but did not have lower terpene concentrations. Decreases of terpene production have been reported when leaf nutrient concentrations increase (Son et al. 1998). Kainulainen et al. (1996) observed that N fertilization had no effect on monoterpene concentrations in growing needles of Pinus sylvestris, but in mature needles, N fertilization significantly decreased concentrations of some individual and total monoterpenes. There also are reports of no relationships at

8 J Chem Ecol (2010) 36: Table 2 Foliar contents of aromatic, cyclic, and non-cyclic monoterpenes (μg g 1 ) detected in the studied native and alien species in Oahu (Hawai i). The species lacking detectable amounts of aromatic and cyclic monoterpenes are shown in Appendix 1. A = alien; N = native Species Origin Ageratina adenophora A p-cymene 340 ± 60 Pinene 27.3 ± 11.2 Camphene β-pinene 14 ± 12 Δ 3 -Carene 18.8 ± 5.9 Limonene 91 ± 12 Thymoquino ne 331 ± 105 Bornyl acetate 1060 ± 172 Terpinene 485 ± 93 Endo-borneol 30.6 ± 3.7 E- Ocimene 20 ± 7 E-β- Ocimene 11 ± 2 Linalool 21 ± 6 Cheirodendron trigynum N Thujene 6.3 ± 3.1 Phellandrene 130 ± 70 Pinene 3500 ± 1100 Sabinene Camphene 30 ± 13 β-pinene 673 ± 533 β-terpinene 165 ± 67 Terpinolene 13 ± 5 Campholene aldehyde 36 ± 20 Terpinene 5 ± 2 Myrcene 163 ± 66 Cinnamomum burmannii A p-cymene 220 ± 100 Camphen e 67 ± 21 β-pinene 910 ± 260 Δ 3 - Carene Thymoquino ne 45 ± 9 E- Ocimene 49.1 ± 17 1,8-Cineole 261 ± 72 γ- Terpinene 3.7 ± 2.6 Terpinolene 8 ± 2 Terpinen-4-ol 6 ± 2 Terpineol 79 ± 22 Terpinolene 133 ± 39 Myrcene 72 ± 24 E-Ocimene 3.6 ± 1.7 L-3,7-dimethyl-1,3,7-Octatriene 21 ± 7 n. id. monoterpene 10 ± 5 Eucalyptus robusta A p- Cymene 77 ± 11 Pinene 330 ± 150 Fenchene 22 ± 5.67 Camphene 14 ± 11.8 Phellandrene 79 ± 24 Limonene 33 ± 10 β- Phellandr ene 5.7 ± 1.4 Campholene aldehyde 6 ± 1 E- Pinocarveol 278 ± 36 3-Cyclohexen- 1-ol. 4-methyl- 1-(1- methylethyl)- 7 ± 2 p-mentha triene 13 ± 2 Terpineol 41 ± 17 Z-p-Mentha-1(7),8-dien-2-ol 6 ± 6 Bornyl formate 15 ± 2 Endo-fenchol 35 ± 7 Endo-borneol 74 ± 16 E-Ocimene 6 ± 1 E-Linalool Oxide 5.1 ± 1.6 Heliocarpus americanus A Camphene 0.30 ± 0.22 Lantana camara A Thujene 4.1 ± 0.4 Phellandrene 16 ± 3 Pinene 105 ± 10.7 Sabinene 90 ± 9 Camphene 43 ± 5.53 Δ 3 -Carene 71.5 ± 7.8 Limonene 66 ± 14 l-camphor 61 ± 9 Terpineol 27 ± 4 Terpinene 4.7 ± 1.9 Terpinolene 14 ± 4 E- Ocimene 18 ± 4 Linalool 31 ± 7 Mangifera indica A Pinene 10.3 ± 0.7 f-phellandrene 5 ± 0 β-phellandrene 30 ± 1 E-Ocimene 1.3 ± 0.5 E-β-Ocimene 3.4 ± 1.3 Melaleuca quinquenervia A Thujene 6.1 ± 1.5 Pinene 746 ± 155 Camphene 12 ± 3.85 β-pinene 188 ± 22 α - Terpinene 79 ± 15 1,8-Cineole 5963 ± 605 γ-terpinene 85 ± 17 Terpinolene 9 ± 4 Isopulegol 37 ± 1 3-Cyclohexen-1-ol. 4- methyl-1-(1- methylethyl)- 15 ± 2 Terpineol 2155 ± exo-2- Hydroxycineole 17 ± 3 Myrcene 32 ± 22 E-Ocimene 11 ± 8 Linalool 82 ± 25 Melicope clusiifolia N p-cymene 9 ± 5 Pinene 3661 ± 123 Camphene 15 ± 8.43 β-pinene 57 ± 33 Limonene 31 ± 15 Z Linalool oxide 33 ± 19 Campholene aldehyde 98 ± 57 E-Pinocarveol 18 ± 11 E-Verbenol 52 ± 30 Myrtenol 7 ± 3 Verbenone 14 ± 8 Bornyl acetate 7 ± 3 Endoborneol 6 ± 1 Myrcene 22 ± 4 E-β-Ocimene 4.8 ± 0.5 Linalool oxide 56 ± 26 Linalool 18 ± 8 Geranyl acetate 27 ± 15 Melicope peduncularis N Pinene 13 ± 6 (1R)-E-Isolimonene 13 ± 2 Limonene 149 ± 113 β-terpinene 39 ± 17 p-mentha triene 6 ± 3 E-Carvyl acetate 8 ± 6 E-Ocimene 1.6 ± 0.2 p-mentha-e-2,8-dien-1-ol 9 ± 7

9 218 J Chem Ecol (2010) 36: Metrosideros macropus N Sabinene 13 ± 5 Metrosideros polymorpha N E-Ocimene E-β-Ocimene 56 ± 17 Myrsine sandwicensis N Limonene 1 ± 1 Myrcene 1.8 ± 1.0 Persea americana A Phellandrene 6.4 ± 0.2 Pinene 113 ± 51 Sabinene 278 ± 27 β-pinene 137 ± 76 1,8-Cineole 77 ± 9 Pimenta dioica A p-cymene 0.13 ± 0.11 Eugenol 4177 ± 1950 Thujene 9.9 ± 8.1 Phellandrene 36 ± 3 Pinene 26 ± 15 Myrcene 9 ± 8 Pluchea carolinensis A Pinene 4 ± 68 Psidium cattleianum A Pinene 26 ± 21 Schinus terebinthifolius A Thujene 75 ± 9 Phellandrene 730 ± 73 Pinene 1115 ± 141 Sabinene 406 ± 49 Syzygium cumini A Phellandrene 2.6 ± 0.3 Pinene 110 ± 20 Fenchene 1.6 ± 0.65 β-pinene 65 ± 36 l-phellandrene 27 ± 3 Syzygium sandwicensis N Camphene 12 ± 1 E-β-Ocimene 523 ± 120 L-3,7-dimethyl-1,3,7-Octatriene 68 ± 6 β-pinene 1.2 ± 1.0 β-thujene 2544 ± 286 Limonene 167 ± 17 E Sabinene hydrate 2.3 ± 0.2 Myrcene 11 ± 3 Δ 3 -Carene 3.13 ± 2.6 1,8-Cineole 133 ± 77 γ- Terpinene 15 ± 12 Terpinolene 64 ± 52 Terpinene 84 ± 21 Myrcene 53 ± 4t E-β-Ocimene 17 ± 2 Terpinolene 5.0 ± 1.9 Terpineol 6 ± 2 Terpinene 107 ± 10 Terpinen-4-ol 0.17 ± 0.14 Terpinolene 0.2 ± 0.1 Terpineol 6 ± 5 Myrcen e Terpinolene 30 ± 25 E-Ocimene 1.5 ± 0.5

10 J Chem Ecol (2010) 36: Table 3 Foliar cyclic sesquiterpene contents (μg g 1 ) in native and alien species in Oahu (Hawai i). Species without detectable sesquiterpene pools are listed in Appendix 1. A = alien; N = native Species Origin Ageratina adenophora A Sabinyl acetate E- Bergamotene β-funebrene Bergamotene Longipinene Zingiberene Bicyclogermacrene β-bisabolene β-sesqui phellandrene Cis-Bisabolene Cadina-1,4-diene γ-curcumene Bisabolol E- Caryophyllene E-β-Farnesene σ-nerolidol Nerolidol (5),6-Guaiadiene Calarene Cheirodendron trigynum N Copaene β- Caryophyllene 7+2 Humulene Zingiberene Eremophilene β-sesqui phellandrene Germacrene B Cycloisolongifol- 5-OL (sesqui) Dehydroaromadendrene 7+2 (3E,5E,8Z)-3,7,11- Trimethyl-1,3,5,8, 10-dodecapentanene Cinnamomum burmannii A Bicyclogermacrene E Bisabolene γ Elemene Spathulenol Caryophyllene oxide Guaiol Isospathulenol 8+2 Eudesmol Eucalyptus robusta A Alloaroma-dendrene Globulol Heliocarpus americanus A Cubebene 6+1 β-elemene Cis-Bisabolene β-selinene Selinene Caryophyllene oxide γ-gurjunene Lantana camara A Copaene β- Caryophyllene Humulene Zingiberene β-cubebene Bicyclo-germacrene Germacrene A Gurjunene E Bisabolene 5+2 Mangifera indica A Humulene β-cubeben Gurjunene Germacrene B γ-curcumene Melaleuca quinquenervia A Copaene Gurjunene β- Caryophyllene Alloaromadendrene Aromadendrene Humulene β-selinene Dehydro aromadendrene Selinene Amorphene Globulol Veridiflorol Ledol Copaene γ-eudesmol Eudesmol β-eudesmol Amorphene Palustrol Caryophyllene oxide Melicope clusiifolia N Z-Caryophyllene Aromadendrene Humulene Aristolene β-selinene γ-cadinene Epiglobulol Spathul enol Caryophyllene oxide Isoledene β-eudesmol Globulol Calarene Eudesmol Melicope peduncularis N Copaene β-guaiene 9+6 Humulene Eremophilene Germacrene B Spathulenol Caryophyl lene oxide Guaiol E- Caryoph yllene 6+5 γ-eudesmol Calarene Metrosideros macropus N Humulene E-Caryophyllene Eudesmol β-eudesmol Bulnesol Metrosideros polymorpha N Copaene Epi- Bicyclosesqui phellandrene γ-gurjunene β-elemene Humulene Isopropyl-5- Bicyclo[4.4.0]Dec-1-en Junipene γ-selinene β-selinene Selinene Gurjunene 8+4 γ- Cadinene Δ- Cadinene Muurolene Caryophyllene oxide 9+3 Metrosideros rugosa N Cubebene Ylangene Copaene β- Cubebene Aromadendrene Humulene Isopropyl-5- Bicyclo[4.4.0]Dec-1-en Amorphene Gurjunene Eremophilene γ-cadinene Muurolene Germacrene B Ledol Δ-Selinene T-Muurolol (3S, 4R, 5S,6R,7S)-Aristol-9-en- 3-ol 8+3 Cadinol Cadina-1,4- diene γ-gurjunene

11 220 J Chem Ecol (2010) 36: Metrosideros tremuloides N Humulene Bicyclo-germacrene 9+16 E-Caryophyllene Myrsine lessertiana N Myrsine sandwicensis N Copaene γ-muurolene Cubebene (3Z)-Cembrene A Ylangene Spathulenol Copaene Bergamotene 8+2 β- Caryophyllen e Caryophyllene oxide Alloaromadendrene Alloaromadendrene T- Muurolo l Aromadendrene Humulene Humulene Isopropyl-5- Bicyclo[4.4.0]Dec-1-en 8+2 γ-cadinene β-cubebene Bicyclogermacrene γ- Seline ne Aristolene Amorphene β-selinene Gurjunene β- Cubebene Muurolene Selinene Spathulen ol Caryophyllene oxide Isoledene Cadinol Calarene Cadina-1,4-diene γ-gurjunene T-Muurolol Junipene Persea americana A D β-cubebene Pimenta dioica A Ylangene β-elemene Aromadendrene Humulene Caryophyllene oxide Calarene 9+4 γ-gurjunene T-Muurolol Pluchea carolinensis A γ-gurjunene Selinene Gurjunene Psidium cattleianum A Cubebene Ylangene Copaene β-maaliene Alloaromadendrene Aromadendrene Humulene Epizonare Germacrene -D β-selinene Selinene Amorphene γ-cadinene E-γ-Bisabolene Muurolene Germacrene B Caryophyllene oxide Veridiflorol Valencene Germacrene- 6,10,11,11-Tetramethyl- tricyclo[ (2,3)undec- 1(7)ene E-β- Farnesene Calarene γ- Curcumene γ- Gurjunene Bisabolol Juniper camphor Psidium guajava A Ylangene Copaene β-maaliene β- Caryophyllene Alloaromadendrene Aromadendrene Humulene β-selinene Selinene Amorphene γ-cadinene E-γ- Bisabolene Muurolene Caryophyllene oxide Veridiflorol Valencene ,10,11,11-Tetramethyl-tricyclo[ (2,3)undec-1(7)ene Calarene γ-curcumene Bisabolol E-β-Farnesene Germacrene B Rubus rosifolius A Cubebene Ylangene Copaene β-cubebene Amorphene γ-cadinene β-copaen-4 ol ,10,11,11-Tetramethyl-tricyclo[ (2,3)undec-1(7)ene Copaenol T-Muurolol caryophylla-3,8(13)-dien -5β-ol 9+5 Schinus terebinthifolius A Copaene β-elemene Humulene Bicyclo-germacrene Δ-Elemene Germacrene B E-Caryophyllene Syzygium cumini A Copaene 6+2 β-elemene Guaiene Humulene Aristolene Germacrene A γ-cadinene Δ-Elemene Muurolene Germacrene B Valencene ,10,11,11-Tetramethyl- tricyclo[ (2,3)undec-1(7)ene Isoledene Calarene Globulol E-Caryophyllene Syzygium sandwicensis N Cubebene Copaene Aromadendrene Humulene β-cubebene Gurjunene Δ-Guaiene Ledol Isoledene Cadina-1,4-diene E-Caryophyllene

12 J Chem Ecol (2010) 36: Table 4 Mean values (SE) of the concentrations of the most abundant terpenes and their ratios to key nutrient contents and photosynthetic capacity per dry mass (A mass ) in relation to sampling site, species origin (native or alien) and soil type. P-values indicate the results of general linear models. OLS-Ordinary least squares regression (see Methods ) Trait Model Site 1 Origin Soil 2 Ta Wi HV SLH P-value Native Alien P-value Inc Oxi Ult Inc_T Moll P-value μg g 1 OLS (30.6) (51.3) (201) (42.0) (46.0) (86) (0) (48) (32.1) (69.0) (49.9) Humulene μg g 1 OLS (0.65) (58) (54.8) (9) (41.9) (24.1) (32.0) (48) (0.65) (13.6) (11.7) Caryophyllene oxide μg g 1 OLS (1.7) (31.7) (5.0) (17.8) (22.7) (5.6) (0) (22.7) (1.8) (49.2) (39) Myrcene μg g 1 OLS (3.42) (6.5) (3.3) (2.0) (4.7) (2.4) (2.8) (4.9) (3.6) (8.1) (1.2) p-cymene μg g 1 OLS (18.7) (0.36) (0) (7.0) (0.3) (10.6) (0) (0.26) (19.7) (19.2) (0.02) Phellandrene μg g 1 OLS (43.6) (5.2) (45.5) (3.3) (3.7) (30.2) (0.4) (21.9) (45.8) (0) (4.5) Pinene μg g 1 OLS (7.6) (198) (69.2) (71) (142) (35.4) (18.3) (144) (7.9) (177) (3.2) Camphene μg g 1 OLS (33.7) (1.4) (2.7) (1.6) (1.0) (18.6) (0) (1.5) (35.4) (3.8) (0) β-pinene μg g 1 OLS (6.5) (26.9) (0) (17.1) (11.2) (6.2) (10.8) (19.2) (6.8) (46.9) (0.15) Total monoterpene μg g 1 OLS (161) (197) (313) (904) (101) (304) (84.1) (197) (169) (2287) (558) Total sesquiterpene μg g 1 OLS (454) (391) (628) (313) (202) (297) (1226) (346) (478) (1664) (84.8) Total terpene μg g 1 OLS ( (660) (465) (875) (1488) (227) (367) (1310) (470) (640) (3942) (642) Total monoterpene/n μg Terp mg 1 N OLS (5.8) (13) (15.4) (83) (9.3) (13.3) (43) (11.5) (6.0) (216) (41) Total monoterpene/p μg Terp mg 1 P OLS (120) (219) (201) (2347) (158) (700) (87.6) (179) (107) (160) (1239) Total sesquiterpene/n μg Terp mg 1 N OLS (14.6) (28.0) (34.0) (57.3) (19.5) (23.8) (61.7) (23.1) (15.3) (156.9) (6.2) Total sesquiterpene/p μg Terp mg 1 P OLS (199) (545) (505) (1613) (361) (540) (1263) (404) (210) (4440) (188)

13 222 J Chem Ecol (2010) 36: Trait Model Site 1 Origin Soil 2 Ta Wi HV SLH P-value Native Alien P-value Inc Oxi Ult Inc_T Moll P-value Total terpene/n μg Terp mg 1 N OLS (20) (32) (45) (138) (19) (29) (65.9) (28.5) (20.9) (372) (47.1) Total terpene/p μg Terp mg 1 P OLS (289) (603) (633) (3913) (409) (1180) (1352) (470) (302) (10590) (1427) 3 Total monoterpene/a mass OLS (1300) (2930) (5233) (15569) (2376) (4742) (557) (3062) (1386) (29246) (0) 3 Total sesquiterpene/a mass OLS (2068) (13888) (8866) (11053) (11357) (4779) (8137) (11265) (2201) (21841) (0) 3 Total terpene/a mass OLS (3218) (14277) (13581) (26305) (11719) (9031) (8684) (12199) (3448) (50450) (0) 1 Ta - Tantalus, Wi - Wiliwilinui, HV Hahaione Valley, SLH Saint Louis Heights (Table 1) 2 Inc Inceptisols (mountainous soils), Oxi - Oxisols, Ult - Ultisols, Inc_T Inceptisols (Tantalus), Moll - Mollisols Significant differences (P<0.05) are highlighted in bold 3 Amass = μmols CO 2 g 1 soil s 1 Total leaf terpene concentration (μg g -1 ) Native Low invasiveness Moderate-high invasiveness High invasiness BMT CMT CST NCMT NCST TMT TST TT Terpene type all between soil nutrients and terpene contents (Heyworth et al. 1998). Our results support the nutrient driving synthesis hypothesis, which expects higher nutrient availability to translate into higher carbon fixation and activity of the enzymes involved in isoprenoid production (Harley et al. 1994; Litvak et al. 1996). Other studies have reported a significant and positive relationship between leaf terpene content and N availability in Pinus halepensis (Kainulainen et al. 2000), and NPK fertilization has shown to increase terpene contents in Chrysanthemum boreale (Lee et al. 2005). On the other hand, our results also support the modified EICA related hypothesis that suggests that alien success may be favored by an increase in the concentrations of less costly defenses such as terpenes that may be more toxic to generalist herbivores (Joshi and Vrieling 2005; Stastny et al. 2005), as has been observed in some previous studies (Johnson et al. 2007). Terpene Content and Success of Aliens The results suggest that alien success may be related to higher levels of leaf terpene content that can have protective effects in response to environmental stress and/or prevent the attack of generalist herbivores and pathogens. The possible role of terpenes as cause of alien success due to herbivorism protection, however, should be taken with caution because the few studies that have examined herbivory pressure in Hawai i are inconclusive. For example, a study by DeWalt et al. (2004) found little fungal and insect damage on one invader; however, Joe and Daehler (2008) found significant slug damage on several rare native species. Terpenes may produce advantages by other mechanisms, e.g., overall higher terpene production and accumulation in alien species could be involved in allelopathic or protective mechanisms b a ab ab ab b a ab ab a a Fig. 2 Foliar benzenic monoterpene (BMT), cyclic monoterpene (CMT), cyclic sesquiterpene (CST), non-cyclic monoterpene (NCMT), non-cyclic-sesquiterpene (NCST), total monoterpene (TMT), total sesquiterpene (TST), and total terpene (TT) concentrations (μg g 1 )in native and alien species grouped according to their invasiveness index. Different letters indicate statistically significant differences at P<0.05 among species groups with differing invasiveness index b