Field comparison of ecophysiological traits between an invader and a native species in a Mediterranean coastal dune

1 This is the peer-reviewed version of the article accepted for publication in PLANT PHYSIOLOGY AND 1 BIOCHEMISTRY Volume 146: 278-286. 2019, which has been published in final form at: 2 doi.org/10.1016/j.plaphy.2019.11.032. 3 Field comparison of ecophysiological traits between an invader and a native 4 species in a Mediterranean coastal dune 5 6 María Zunzunegui1*, Elena Ruiz-Valdepeñas1, Maria A. Sert2, Mari Cruz Díaz-Barradas1, 7 Juan B. Gallego-Fernández1 8 9 1Departamento de Biología Vegetal y Ecología, Universidad de Sevilla, Apartado 1095, 10 41080 Sevilla, SPAIN 11 2Universidade Estadual de Maringá, Departamento de Biología, Maringá, BRASIL 12 * Corresponding author E-mail address: [email protected] (telephone: +34 954557070) 13 14 ABSTRACT 15 Photochemical efficiency, gas exchange, leaf water potential and pigment and free proline 16 content of Oenothera drummondii subsp. drummondii Hook (an invasive non-native 17 species) and Achillea maritima (L.) Ehrend. & Y.P. Guo, (an ecologically similar native 18 species) were explored to understand the success of invasive non-native species in 19 Mediterranean coastal dunes of southwest Spain. We have conducted a field study during 20 a complete annual cycle, comparing both species. Fifteen pairs of neighbouring plants of 21 the two study species of similar size were selected and measured seasonally. The results 22 show that in spring and summer, assimilation rates of O. drummondii were significantly 23 higher than those of the native, A. maritime, even though the native species had higher 24 photochemical efficiency. Additionally, the non-native species presented better water 25 content regulation than the native one, surely related to better water use efficiency and 26 may be linked to greater root development. The differences in leaf dry matter content 27 values for both species might indicate a different strategy of resource use; with A. 28 maritima displaying a more conservative strategy and O. drummondii presenting a rapid 29 resource acquisition and use strategy as predictors of rapid growth and soil fertility. We 30 conclude that O. drummondii utilizes light, water and probably nutrients more efficiently 31 than the native A. maritima and suffers lower stress in Mediterranean coastal dunes where 32 water availability is reduced (44 mm from May to October in the study area) and light 33 radiation levels are high. 34 35 Keywords: Achillea maritima; Invasiveness; Leaf water potential; Mediterranean-type 36 ecosystems; Oenothera drummondii; Photosynthesis. 37 2 Introduction 38 39 Coastal dunes are complex and dynamic ecosystems where vegetation is subjected to 40 many stressors including salinity, episodic overwash, water stress, high temperature, high 41 winds, sand burial, substrate instability, low field capacity, and nutrient scarcity as the 42 most common abiotic stressing factors that influence plant community assembly 43 processes in dune systems (Hesp, 1991; Maun, 2009). Additionally, physiological plant 44 activity in Mediterranean-type ecosystems is limited during summer by high 45 temperatures, water scarcity and high radiation levels (Cowling, et al. 2005; Godoy et al., 46 2011), but it is also limited in winter through the combination of low temperatures with 47 high radiation (Oliveira and Peñuelas, 2004; Flexas et al., 2014). Therefore, in the coastal 48 dunes of the Mediterranean type climate, these stressors reinforce each other intensifying 49 the vegetation stress level. As a consequence of the notable selection pressure imposed 50 by these environmental conditions, the coastal dune vegetation has a high degree of 51 specialization (Novoa et al., 2012), particularly in Mediterranean type climate. In beach 52 and embryo dunes frequent occurrence of intense disturbance events provide invasive 53 species with the opportunity to colonize novel areas (Lortie and Cushman 2007), 54 additionally, in inland dunes, intermediate levels of disturbance and stress can also offer 55 good conditions for the settlement of invasive species (Carboni et al., 2010). That is, the 56 patterns of species invasions on coastal dunes depend on the environmental conditions 57 and the species involved (Gallego-Fernández et al., 2019). 58 Coastal dune environmental conditions impose adaptations to plants that stablish in 59 these areas. For instance, to withstand salt, plants increase leaf thickness and the degree 60 of succulence, while to cope with burial they produce larger seeds and have stolons or 61 rhizomes (Hesp, 1991). Water-limited resources are a primary stressor in coastal dune 62 ecosystems; therefore, many coastal dune plants have adaptations to resist dryness. The 63 adjustments to dryness occur at different plant levels: physiological, reproductive, 64 morphological at the leaf, roots, or the whole plant structure. At the leaf level, for 65 example, plants can adjust leaf rolling, thickness, orientation, hairiness, size or epicular 66 wax. At physiological levels, plants can have osmotic adaptations to increase water use 67 efficiency or different photosynthetic pathways such as C4 and CAM (Hesp, 1991). 68 As a result of the high specialization grade imposed by these harsh environmental 69 conditions, these ecosystems support many threatened and endemic species which are 70 especially sensitive to invasions by non-native plants. One of the greatest threats of 71 3 natural habitats around the world is the introduction of exotic species (Sax and Gaines, 72 2003) and on coastal dune ecosystem in particular, this has been assessed as high and still 73 increasing (Millennium Ecosystem Assessment, 2005; Novoa et al., 2012), mainly due to 74 the frequent stressor and the existence of open patches free of plant competition (Carboni 75 et al., 2010; Antunes et al., 2018). 76 The success of invasive plants on novel environments depends on several factors, 77 such as the adaptive plasticity of their morphological or physiological traits and the 78 environmental conditions of the new area. Alternatively, numerous studies (Oduor et al., 79 2016; Liao et al., 2016) support that pre-adaptation or rapid adaptive evolution to new 80 ecological niches could be as important as plasticity to explain the ability of invasive 81 plants to persist and expand in new-introduced habitats. These two processes would not 82 be mutually exclusive. Thus to maximize fitness in response to new environmental 83 conditions a combination of plasticity and rapid genotypic adaption could occur. 84 According to Liao et al. (2016) plasticity plays an important role in invasiveness, but also 85 local genotypic variation in plasticity does. 86 Besides, also the biological interactions (such as plant-plant competition or plant87 herbivore interaction) have an ecological impact on original communities and in the 88 ecosystem function which is crucial for the success of plant invasion (Ehrenfeld, 2010; 89 Bottollier-Curtet et al., 2013). Invasive plants commonly compete so effectively in new 90 ecosystems that they displace native species and alter ecosystem function and ecosystem 91 services. Van Kleunen et al. (2010) studied what functional traits were associated with 92 invasiveness (physiology, allocation, growth, size or fitness), considering invasiveness as 93 the capacity of plants to spread through other biota and ecosystems. They found that 94 invasive non-native species had higher values for traits related to performance than non95 invasive species. The question of what kinds of traits promote plant invasiveness is of 96 utmost significance for understanding plant success in general, and particularly for 97 understanding the mechanisms of non-native plant invasions. However, studies that 98 suggest that invasive species have advantages over native ones have had irregular results 99 and moreover, the available data of the positive effect of physiological traits on invasive 100 species' fitness are inconsistent. 101 Nonetheless, invasive species expected to have physiological traits (such as higher 102 photosynthetic rates or better stomatal control) than allow them to take advantage of a 103 better physiological status and overcome natives. A comparative study of physiological 104 4 plant activity in co-occurring invader and native species is a useful tool to improve 105 understanding of the invasiveness of exotic plants. Plant ecophysiological characteristics 106 must be well adapted to their local environmental conditions (Ackerly et al., 2000) so 107 that, the physiological response of plants to local conditions can play a fundamental role 108 in species’ ecological distribution (Körner and Diemer, 1987; Brodersen et al., 2008). 109 Physiological adjustments allow species to maintain their fitness under different 110 environmental conditions (Becklin et al., 2016). Among these ecophysiological 111 characteristics, photosynthesis and water status-related traits have been proven to have a 112 considerable influence on the invasive success of non-native species (McDowell, 2002). 113 Oenothera drummondii subsp. drummondii is a non-native invasive species of 114 coastal dunes around the world; native to coastal dunes of the Gulf of Mexico and in the 115 last century has colonized coastal dunes all over the world (Dietrich and Wagner, 1988). 116 Nowadays it has spread over coastal areas of different continents, being considered 117 invasive in Spain, Israel, China and Australia and naturalized in many places of the world, 118 shifting to regions with different climatic characteristics (Dietrich 2000; Heyligers 2008; 119 Campos and Herrera 2009; Xu et al. 2012, Dufour-Dror 2013; García de Lomas et al. 120 2015). 121 In coastal dunes of southwestern Spain, O. drummondii has produced a strong 122 impact on native communities by modifying the composition and abundance of the 123 species (García de Lomas et al., 2016; Gallego-Fernández et al., accepted). To understand 124 some of the causes of the success of this non-native species we have compared our 125 observations of O. drummondii with a species of similar characteristics that has been 126 displaced after the invasion and that used to be highly abundant, Achillea maritima. Both 127 are small-sized plants (40-50 cm tall) with reproduction mainly by seeds. The purpose of 128 this study is to establish whether the success of O. drummondii can be explained in part 129 by differences from native species in the physiological response to Mediterranean coastal 130 dune environment. To answer this question, we compared the physiological performance 131 of both species measuring the photochemical efficiency, gas exchange, leaf water 132 potential and free proline and pigment content under field conditions over the whole year. 133 All these ecophysiological variables have proven to have a considerable influence in the 134 invasive success of the non-native species (McDowell, 2002) while free proline in leaves 135 has been widely proved to accumulate as a response to numerous stressors (such as 136 drought or cold temperatures). Additionally, weather conditions also induce changes in 137 5 the pigment content (Hoffmann and Parsons, 1997; Gratani, 2014, Zunzunegui et al., 138 2016). We hypothesized that O. drummondii would exhibit a high capacity to adapt to 139 Mediterranean environmental conditions presenting higher photochemical efficiency, 140 assimilation rates and water use efficiency than the native species 141 142 Material and methods 143 144 Study site 145 The study was accomplished on a coastal dune system located in the protected area 146 “Marismas del Odiel” close to Huelva city, SW Spain (37º09'N, 6º54'W). The climate is 147 Mediterranean with hot summer; the type is Csa, according to the Köppen-Geiger 148 classification (Peel et al., 2007). The mean annual temperature is 18.1 °C while the mean 149 minimum and maximum temperatures are 13.3 and 21.7° C, respectively. Average annual 150 rainfall is 467 mm, with a pronounced drought period in summer (data from Agencia 151 Estatal de Meteorología at Huelva Meteorological Station, 30-year record from 1971 to 152 2000). The year of the study, with precipitation of 296 mm, was 36% dryer than the annual 153 average (Fig. 1), with remarkably low precipitation during the warmest period of the year 154 in which the accumulated rainfall from May to October was 44 mm. 155 Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Precipitation (mm) , Relative Humidity (RH,%) 0 20 40 60 80 100 Temperature (ºC) 0 5 10 15 20 25 30 35 40 45 50 Vapour pressure deficit (VPD, KPa) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 RH 2013 2014 Precipitation T max T min VPD 156 157 158 Fig. 1 Monthly precipitation, daily maximum and minimum mean temperatures and air 159 relative humidity during the study period (2013-14 hydrological cycle). Arrows indicate 160 sample periods. 161 162 6 Study species 163 164 Oenothera drummondii subsp. drummondii Hook. (Onagraceae), is a short-lived leaf 165 perennial species. The main stems are erect to procumbent while the basal side stems are 166 prostrate or ascending to about 50 cm in height, with a strong taproot. Flowers are self167 compatible, outcrossing and pollinated by hawkmoths in their native habitat (Wagner et 168 al., 2007). The species reproduces and spreads through small and numerous seeds. In its 169 native range area, the species grows in coastal dunes with humid subtropical and wet 170 tropical climates (Cfa, Aw, Am) according to the Köppen-Geiger classification (Peel et 171 al., 2007). The first records of O. drummondii in the southwest of the Iberian Peninsula 172 date from 1957 (Silvestre, 1980) and in the study area it was registered for the first time 173 in 1996 (García Mora com. pers). 174 Achillea maritima (L.) Ehrend. & Y.P. Guo is a pioneering herbaceous perennial 175 species, with 20-40 cm of height, from the Asteraceae family (Ehrendorfer and Guo, 176 2005). This species grows in sand coast dunes from Europe and Morocco and exerts a 177 stabilizing action on the sandy soils (García Novo and Merino, 1993). In the Iberian 178 Peninsula, the species is in regression, having disappeared, or almost, in some regions 179 (Silván and Campos, 2002; Mayoral, 1999) mainly because of the loss of its natural 180 habitat due to anthropic pressure. Thick white fuzz covers both the stems and the small 181 oval, alternate leaves. The globose yellow flowers are entomophilous and visible from 182 June to September. The selection of this species was based on the fact that it is one of the 183 most abundant native plants in the studied site and has similar functional traits to the 184 invasive species. Moreover, Gallego-Fernandez et al. (2019) found evidence that this 185 species is out-competing A. maritima since in dunes invaded by O. drummondii the cover 186 of A. maritima is 91% lower than in non-invaded dunes. 187 188 Experimental design 189 190 Field measurements were made in a 50 x 50 m plot located inside the dune zone. In this 191 plot, we randomly selected 15 pairs of neighbouring mature plants of Oenothera 192 drummondii and Achillea maritima of similar size. All pairs were separated more than 2 193 meters from each other. The measurements were carried out with a seasonal periodicity 194 during an annual cycle: November (autumn, with mild temperatures and when the first 195 rains after the summer drought allow plants to recover water status), February (winter, 196 the period of the wet season with the coldest month), April (spring, period with mild but 197 7 increasing temperatures and at the end of the rainy season) and July (summer, with the 198 highest temperatures and radiation coupled with drought). 199 Photochemical efficiency of photosystem II: Chlorophyll fluorescence kinetics was 200 determined in the field on healthy fully expanded leaves using a pulse-amplitude 201 modulation technique through a portable fluorometer (mini-PAM, Walz, Effeltrich, 202 Germany). Fluorescence was excited by a saturating pulse of red light (approximately 203 3000 μmol m-2s-1 for 0.8 s) from a diode (type H-3000 LED, Stanley) attached to a fibre 204 optic. Leaves were exposed to a weak modulated measuring beam from a LED at a 205 frequency of 600 Hz to determine F0 and F and then to the pulse of saturating red light to 206 assess Fm and F’m. 207 Maximum photochemical efficiency of PSII (Fv/Fm) was measured on dark-adapted 208 leaves (leaves were artificially maintained 20 minutes in darkness by means of leaf-clips, 209 a time interval considered sufficient to complete the reoxidation of all the reaction centres 210 of photosystem II) from the ratio of variable fluorescence to maximum fluorescence as 211 Fv/Fm = (Fm−F0)/Fm, where F0= initial and Fm= maximal fluorescence (Genty et al., 1989). 212 Effective photochemical efficiency of PSII (ΦPSII) was estimated on light-adapted leaves 213 as ΦPSII= (F’m-F)/F’m, where F’m= maximal and F steady-state fluorescence under actinic 214 irradiance (Genty et al., 1989). 215 Three leaves were measured per plant at 9:30-11:30 h solar time (mean values per 216 plant were used for statistical analysis). 217 Gas exchange measurements: Measurement of net photosynthetic or net CO2 218 assimilation rate (An, μmol CO2 m-2s-1), transpiration rate (E, mmol H2O m-2s-1), stomatal 219 conductance rate (gs, mol H2O m-2s-1) and leaf intercellular CO2 concentration (Ci, vpm) 220 were taken on mature leaves during clear sunny days, using portable open system, 221 compact infra-red gas exchange analyser (LCi-Portable Photosynthesis, ADC, UK). From 222 these set of data, we calculated instantaneous water use efficiency (WUEi, mmolCO2 mol223 1H2O) as the ratio of net photosynthesis to transpiration (An/E) indicative of the moles of 224 CO2 assimilated per mol of H2O lost, and instantaneous carboxylation efficiency (An/Ci, 225 molCO2 m-2s-1) as the ratio of net assimilation to leaf intercellular CO2 concentration. 226 Three leaves per plant were measured and the average values per plant calculated. 227 The surface of leaves used for gas exchange measurements was calculated in the 228 laboratory using the image area analyser software Midebmp (Ordiales, Spain, 2000). 229 8 Measurements were made from 8:30 to 10:30 h, (solar time), to avoid possible midday 230 depression of stomatal conductance and to get maximum net photosynthetic rates. 231 Leaf water potential: Plant water status was evaluated through midday leaf water 232 potential (Ψmd) and was determined with a pressure chamber (Manofrigido, Lisbon, 233 Portugal) in the field using terminal shoots which were excised and immediately 234 measured. The readings were made between 12:30 and 14:00 (solar time), when the 235 greatest water deficit of the day occurs and when the minimum values are reached. 236 Leaf parameters: Leaves used and scanned for photosynthesis measurements were 237 dried at 70 °C for 48 h and weighted (Leaf mass area (LMA) was calculated as the ratio 238 of dry leaf mass (Md) to fresh leaf area (g m-2). 239 New, fully expanded, healthy leaves were collected from all plants selected for 240 physiological measurements. Leaves for relative water content (RWC) and leaf dry matter 241 content (LDMC) measurements were kept refrigerated and stored in plastic bags until 242 fresh mass (Mf) was recorded within 3 h. After weight, leaves were then hydrated with 243 distilled water to saturation for 24 h at 5°C in the plastic bags. Leaves were dried with 244 tissue paper to remove any surface water, and then immediately weighed to obtain 245 saturated mass (Ms). Finally, leaf samples were then dried at 70 °C for 48 h and weighted 246 (Md). We estimated RWC (%) as follows: 𝑀𝑓−𝑀𝑑 𝑀𝑠−𝑀𝑑 𝑥100 247 The ratio LDMC (mg g-1), also known as tissue density was calculated as Md/Ms. 248 The rest of the collected leaves were kept in liquid nitrogen in the field and preserved 249 in the laboratory at −24 °C until analysis. Photosynthetic pigments were quantified 250 following Lichtenthaler (1987) on a dry mass basis. Chlorophyll a (Chl a), b (Chl b) and 251 total carotenoid (Car) pigments were extracted with 100 % acetone and determined 252 spectrophotometrically. From these data were calculated the ratio chlorophyll a/b (Chl 253 a/b) and the ratio of total carotenoids to total chlorophylls (Car/Chl), 254 Free proline content was determined colourimetrically by the ninhydrin acid method 255 of Bates et al. (1973). 256 257 Statistical analyses 258 Two-way ANOVAs were used to investigate the effects of species and season 259 (independent factors) on ecophysiological variables (dependent factor). Post-hoc Tukey 260 tests were then used for appropriate pair-wise comparisons. Bivariate correlation tests 261 were made between the different variables. A multivariate principal component analysis 262 (PCA) was performed with the physiological and leaf parameters matrix (21 variables x 263 9 4 seasons x 2 species x 15 plants) to identify which are the variables responsible for the 264 different seasonal response between species. The normality of all measured variables was 265 first checked with the Kolmogorov–Smirnov test. The statistical tests were considered 266 significant at the P < 0.05 level. All statistical tests were made with the software package 267 IBM SPSS Statistics for Windows, Version 24 (Armonk, NY, USA). 268 269 Results 270 271 Photosynthetic rates ranged close to 20 μmol m-2s-1 in the non-native species throughout 272 the year, whereas in the native this variable was seasonal dependent, with pronounced 273 drops in spring and summer (Fig. 2A). O. drummondii assimilation rates were 274 significantly higher than those of A. maritima in spring and summer (P < 0.008), while 275 no differences occurred in autumn and winter. As observed for An, and responding to 276 summer stressful conditions, native plants had lower An/Ci values in spring (P < 0.016) 277 and summer (P < 0.001) than non-native plants (Fig. 2B). Native plants showed lower gs 278 in spring and summer but both species showed similar gs values after the first rains in 279 autumn and winter (Fig. 2C). Non-native species showed greater WUEi than native plants 280 during the drought period (Fig. 2D). 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 16 indicate the variables correlated with PC2. An/Ci: instantaneous carboxylation efficiency, 418 Car: total carotenoid content, Ci: leaf intercellular CO2, Chlt: total chlorophyll content; 419 LDMC: leaf dry matter content, LWP: leaf water potential, WUEi: instantaneous water 420 use efficiency. 421 422 Discussion 423 424 Our main outcome in this study is that in spite of having higher photosynthetic rates in 425 spring and summer and better water status through the year, the non-native species O. 426 drummondii had lower photochemical efficiency than the native, A. maritima. The better 427 ecophysiological performance of O. drummondii appears to be related mostly to capture 428 of resources, such as CO2 assimilation, and water-use strategy. 429 Respecting maximum photochemical efficiency, and considering the optimum 430 values between 0.75–0.85 in normal plants (Bolhàr-Nordenkampf and Öquist, 1993, 431 Maxwell and Johnson, 2000), the Fv/Fm values in A. maritima denote that the native 432 species, with values through the year higher to 0.760, was not under particularly stressing 433 conditions. These data agree with those recorded by Scorce et al. (2019) in the same 434 species in a coastal dune system in Italy. In contrast, ΦPSII was affected by the 435 meteorological conditions, pointing out the species underwent certain dynamic 436 photoinhibition, especially in winter when the lowest values were recorded. When 437 compared with the invasive species, A. maritima was photochemically more efficient. 438 Leaf pubescence in A. maritima (Ciccarelli et al., 2009), an adaptation of plants to the 439 Mediterranean climate, reflect photosynthetically active radiation and provides protection 440 against UV-BA radiation and inhibits the reduction in photochemical efficiency 441 (Grammatikopoulos et al., 1994; Bisba et al., 1997). Pubescence increases the reflectance 442 of the leaves, improving plant energy balance and therefore diminishing the risk of 443 photoinhibition (Díaz Barradas et al., 1999; Zunzunegui et al., 1999; 2005). So the higher 444 photochemical efficiency values found in A. maritima agree with higher Fv/Fm values in 445 pubescence leaves as it has been found in other studies (Savé et al., 2000). 446 The fact that the lowest photochemical efficiency values were measured in winter 447 in the non-native species could be due to the tropical origin that makes it more sensitive 448 to low temperatures as it has been proven in other species with a tropical origin 449 (Zunzunegui et al., 2005). In these species, the risk in photoinhibition might be higher in 450 winter than in summer, as lower temperature might inhibit the photosynthetic electron 451 17 chain. Nevertheless, despite the lower photochemical efficiency in the invasive species, 452 the photosynthetic rates recorded for this species in spring and summer, were higher than 453 for the native one. This, therefore, indicates the sensitivity of O. drummondii to cold but, 454 simultaneously that it must be a species of great photosynthetic capacity because it is able 455 to overcome the native species despite its lower photosynthetic efficiency. That fact 456 would help to explain a 91% lower cover of A. maritima in dunes invaded by O. 457 drummondii (Gallego et al., 2019) 458 The meteorological data show that the harsh summer meteorological conditions, 459 (high temperature and radiation together with low relative humidity and water 460 availability) represent an important element in regulating the seasonal photosynthetic 461 activity. But above all, these results are an important differentiator of the best competitive 462 capacity of the invasive species. We may argue that increasing VPD (0.34 to 1.22 KPa) 463 from winter to summer caused the decreasing trend of gs and the lower photosynthetic 464 rate of A. maritima in summer; since VPD has been considered the main environmental 465 variable controlling stomatal behaviour and causing the decrease of the photosynthetic 466 rate in Mediterranean woody vegetation (Tenhunen et al., 1987; Tognetti et al., 1998). On 467 the contrary, the spring and summer VPD effect on gs was not patent in the invasive 468 species. 469 Summer conditions affected the variables An and An/Ci in an opposite way to both 470 species; while in the non-native species the figures increased and the highest annual 471 values were registered, in the native plants they decreased, and the lowest values were 472 recorded. Reductions in instantaneous An/Ci suggest the occurrence of a non-stomatal 473 limitation of photosynthesis and some mesophyll limitation on the photosynthesis of 474 studied plants (Silva et al., 2010). Although photosynthesis rate and stomatal conductance 475 have been described to have a linear correlation in response to environmental conditions 476 (Ball et al., 1987) other factors may be acting to increase An, as Rubisco kinetic or 477 mesophyll conductance that could explain that the highest annual An values recorded in 478 O. drummondii were not linked to the highest stomatal conductance values. 479 The high carboxylation efficiency values recorded in O. drummondii in summer 480 were supported by higher photosynthetic activity with no significant variations of Ci while 481 the low values recorded in A. maritima were supported by high Ci values and low 482 photosynthetic rates. 483 18 The WUEi is a major trait of the water economy of plants and of their performance, 484 survival capacity and functioning in arid sites (Damesin et al., 1997; Bacon, 2004). 485 Summer conditions similarly affected WUEi in both species, as shown by the parallel 486 decline in this variable in spring and summer, especially for A. maritima, when the plants' 487 water status decreased to -1.1 MPa. A high evapotranspiration rate in summer was 488 responsible for the reduction in WUEi especially in the case of O. drummondii, where the 489 photosynthetic rate increased during summer. Scorce et al. (2019) also studying A 490 maritima, recorded summer values of Ψmd as low as -2 MPa, Thus the Ψmd values 491 registered in this study are moderately high. The neighbourhood of the sea might provide 492 moisture to these species during the night and early morning, causing the recorded values 493 in LWP were not as negative as in other Mediterranean species. Nonetheless, O. 494 drummondii always displayed higher RWC values along with higher Ψmd values than A. 495 maritima. This fact denoted a different strategy in water use or access and would be 496 indicative of better water status of the invasive species. 497 Under winter conditions both species presented similar photosynthetic rates but 498 significant differences in hydric status. Photosynthetic activity varies with environmental 499 conditions, and although plants can acclimate to these changes, given the tropical origin 500 of the non-native species, lower photosynthetic rates could be expected in the cold season 501 compared with the native one. Leaves can have different photosynthetic rates under the 502 same environmental conditions due to different stomatal conductance caused by internal 503 or external factors. It has been suggested that changes in the response of cell-wall 504 conductance may be an important factor in temperature acclimation (Makino et al., 1994). 505 The high stomatal conductance in O. drummondii leaves along the year could be the 506 underlying factor in its optimal winter response and better water status. 507 Furthermore, the maintenance of high intercellular CO2 concentration (data not 508 shown) associated with the lowest annual photosynthetic rate in O. drummondii suggests 509 the occurrence of non-stomatal limitation of photosynthesis in winter, while in A. 510 maritima, high intercellular CO2 concentration values combined with the highest annual 511 photosynthetic rate recorded suggests the occurrence of stomatal limitation in the native 512 species. Also, high An/Ci values recorded in A. maritima indicate a non-mesophyll 513 limitation on photosynthesis. 514 The highest LDMC and LMA values recorded in the native species leaves in all 515 seasons indicate that this species would be relatively tougher and more resistant to 516 19 physical stresses than O. drummondii, whose leaves have lower LDMC and LMA. Leaf 517 mass area can be interpreted as the cost of light interception at the leaf level (Gutschick 518 and Wiegel, 1988; Poorter et al., 2009). Increases in LMA are often associated with 519 drought exposure and high irradiance (Gratani and Bombelli, 1999). According to these 520 results, higher fitness could be expected in the native A. maritima, and although the 521 species presented significant higher photochemical efficiency in the most stressful period 522 of the year, the highest photosynthetic rate was always measured in the invasive species 523 O. drummondii. On the other hand, these higher photosynthetic rates in the invasive 524 species are consistent with lower LDMC values recorded, since this functional trait 525 indicates the species’ resource use strategy and represents the compensation between 526 assimilation and rapid growth versus efficient conservation of resources (Wilson et al., 527 1999; Hodgson et al., 2011). The results in LDMC values would indicate a different 528 strategy of resource use for both species; with A. maritima displaying a more conservative 529 strategy as a predictor of poor environments and O. drummondii presenting a rapid 530 resource acquisition and use strategy as a predictor of rapid growth and soil fertility. 531 These outcomes would reinforce the concern that should accompany the invasion by this 532 species due to its rapid resource assimilation and growth which promote its expansion 533 (Garnier et al., 2001; Díaz et al., 2004). 534 Pigment study is important from an ecophysiological perspective as it provides 535 information about productivity, stress or limiting nutrients. Across the most stressful 536 seasons of the year, winter and summer, the invasive species has the advantage over the 537 native of its higher chlorophyll content but also higher Chl a/b ratios in autumn and 538 summer and higher Car/Chl ratios in winter. These variables are both representative of 539 stress. The Chl a/b ratio is indicative of the light absorption capacity of photosystem I to 540 II; declines in this variable are indicative of differential degradation of Chl a concerning 541 Chl b. Under stress conditions, a more rapid destruction of Chl a than Chl b might occur; 542 whereas Car/Chl decreases point out to damage in the photosynthetic apparatus, which is 543 expressed by a faster breakdown of chlorophylls than carotenoids (Lichtenthaler and 544 Buschmann, 2001). This higher pigments concentration can be a response to avoid 545 photochemical damage and an increase in reactive oxygen species due to the combination 546 of low temperatures with high radiation in Mediterranean winter. When the incident light 547 intensity is higher than necessary for photosynthesis reactive intermediates can be 548 produced in excess, leading to oxidative damage (Aro et al., 1993). The presence of 549 20 photoprotective carotenoids under this combination of winter stressful conditions is 550 believed to be a feature of the photoprotective function of carotenoid pigments (Havaux 551 and Kloppstech, 2001) and consequently, the invasive species could have better stress 552 tolerance or better maintenance of physiological functions under unfavourable 553 environmental conditions. 554 Principal component analysis established the presence of three groups 555 corresponding to seasons (summer of both species and O. drummondii winter). According 556 to Wilson et al. (1999), LDMC seems to be the best variable for locating plant species on 557 a resource use on the first axis and also as predictors of the different strategies for the 558 invasive and native species on resources use. The accumulation of Chl and Car, the other 559 variables defining this first axis, can be also related to resources used as optimal growth 560 conditions, as well as sufficient nutrients are positively correlated with pigment contents 561 and levels (del Campo et al., 2000; He et al., 2013). In contrast, axis two seems to separate 562 the two species and seasons according to its water use strategy. 563 In summary, even though O. drummondii has a tropical/subtropical origin, our data 564 show that this species is well acclimated to Mediterranean coastal dunes, exhibiting a 565 higher photosynthetic rate and better water performance than the native species A. 566 maritima. We conclude that O. drummondii utilizes light, water and probably nutrients 567 more efficiently than the native A. maritima and suffers lower stress in environments with 568 reduced water availability and high light radiation. Our results seem to indicate that the 569 underlying mechanism that confers O. drummondii its competitive capacity may be its 570 ability in water uptake, which makes it an effective non-native invasive species; the only 571 limiting factor to control its expansion in the Mediterranean coastal dune could be low 572 temperatures. In the study area, removal projects have been carried out to restore invaded 573 dunes (García-de-Lomas et al., 2016, personal observation) and they have proven the 574 difficulties to maintain the population level below an impact threshold. 575 As the authors, Flores-Moreno and Mole (2013), sustain it is evident that all non576 native species are not super plants with high invasive capacity and capable of maintaining 577 a higher fitness than the co-occurring native species in their new ranges. Furthermore, 578 studies comparing non-native and native species performance are contradictory (Daehler, 579 2003; Palacio-López and Gianoli, 2011). However, in the case of O. drummondii our 580 findings suggest that it is particularly successful in the Mediterranean range (including 581 other distribution areas with Mediterranean climate, Csa, where this species is expanding 582 21 such as the coast of Israel and the west coast of Australia). Therefore we would advise 583 managers to take measures for their eradication in the early stages of their appearance in 584 the Mediterranean dune ecosystems since it is extremely successful in these habitats. 585 586 Acknowledgements 587 588 We thank the staff of the Paraje Natural Marismas del Río Piedras y Flecha de El 589 Rompido for facilities to carry out the fieldwork and Denise for her support in fieldwork. 590 Funding was provided by Ministerio de Economía y Competitividad (MINECO Project 591 CGL2015-65058-R cofounded by FEDER). 592 593 References 594 595 Ackerly, D.D., Dudley, S.A., Sultan, S.E., Schmitt, J., Coleman, J.S., Linder, C.R., 596 Sandquist, D.R., Geber, M.A., Evans, A.S., Dawson, T.E., et al., 2000. The 597 evolution of plant ecophysiological traits: recent advances and future directions. 598 Bioscience 50, 979–995. 599 Ain-Lhout, F., Zunzunegui, M., Díaz Barradas, M.C., Tirado, R., Clavijo, A., García 600 Novo, F., 2001. Comparison of proline accumulation in two Mediterranean shrubs 601 subjected to natural and experimental water deficit. Plant Soil 230, 175–183. 602 Antunes, C., Pereira, A.J., Fernandes, P., Ramos, M., Ascensão, L., Correia, O., Máguas, 603 C., 2018. Understanding plant drought resistance in a Mediterranean coastal sand 604 dune ecosystem: differences between native and exotic invasive species. J. Plant 605 Ecol. 11, 26–38. 606 Aro, E.M., Virgin, I., Andersson, B., 1993. Photoinhibition of photosystem II. 607 Inactivation, protein damage and turnover. Biochim. Biophys. Acta 1143, 113608 134. 609 Bacon, M.A., 2004. Water use efficiency in plant biology. Oxford: Blackwell Publishing 610 Ltd/CRC Press. 611 Ball J.T., Woodrow I.E., Berry J.A., 1987. A Model Predicting Stomatal Conductance 612 and its Contribution to the Control of Photosynthesis under Different 613 Environmental Conditions. In: Biggins J. (eds) Progress in Photosynthesis 614 Research. Springer, Dordrecht 615 Bates, L.S., Waldren. R.P., Teare, I.D., 1973. Rapid determination of free proline for 616 water stress studies. Plant Soil, 39, 205–207. 617 22 Becklin, K.M., Anderson, J.T., Gerhart, L.M., Wadgymar, S.M., Wessinger, C.A., Ward, 618 J.K., 2016. Examining Plant Physiological Responses to Climate Change through 619 an Evolutionary Lens. Plant Physiol. 172, 635-649. 620 Bisba, A., Petropoulou, Y., Manetas, Y., 1997. The transiently pubescent young leaves 621 of plane (Platanus orientalis) are deficient in photodissipative capacity. Physiol. 622 Plantarum 101, 373–378. 623 Bottollier-Curtet, M., Planty-Tabacchi, A.M., Tabacchi, E., 2013. Competition between 624 young exotic invasive and native dominant plant species: implications for 625 invasions within riparian areas. J. Veg. Sci. 24, 1033-1042. 626 Bolhàr-Nordenkampf, H.R., Öquist, G., 1993. Chlorophyll fluorescence as a tool in 627 photosynthesis research. In: D.O. Hall, J.M.O. Scurlock, H.R. Bolhàr628 Nordenkamppf, R.C. Legood, S.P. Long (Eds.), Photosynthesis and production 629 in a changing environment: A field and laboratory manual. London: Chapman and 630 Hall. 631 Brodersen, C., Lavergne, S., Molofsky, J., 2008. Genetic variation in photosynthetic 632 characteristics among invasive and native populations of reed canary grass 633 (Phalaris arundinacea) Biol. Invasions 10, 1317–1325. 634 del Campo, J.A., Moreno, J., Rodriguez, H., 2000. Carotenoid content of Chlorophycean 635 microalgae: Factors determining lutein accumulation in Muriellopsis sp. 636 (Chlorophyta). J. Biotechnol., 76, 51–59. 637 Campos, J.A., Herrera. M., 2009. Diagnosis de la Flora alóctona invasora de la CAPV. 638 Gobierno Vasco, Bilbao, 296 pp. 639 Carboni, M., Santoro, R., Acosta, A.T.R., 2010. Are some communities of the coastal 640 dune zonation more susceptible to alien plant invasion? J Plant Ecol. 3, 139–47. 641 Ciccarelli, D., Forino, L.M.C., Balestri, M., Pagni, A.M., 2009. Leaf anatomical 642 adaptations of Calystegia soldanella, Euphorbia paralias and Otanthus maritimus 643 to the ecological conditions of coastal sand dune systems. Caryologia 62, 142– 644 151. 645 Cowling, R.M., Ojeda, F., Lamont, B.B., Rundel, P.W., Lechmere-Oertel, R., 2005. 646 Rainfall reliability, a neglected factor in explaining convergence and divergence 647 of plant traits in fire-prone Mediterranean-climate ecosystems. Global Ecol. 648 Biogeogr. 14, 509–519. 649 23 Daehler, C.C., 2003. Performance comparisons of co-occurring native and alien invasive 650 plants: implications for conservation and restoration. Annu. Rev. Ecol. Evol. Syst. 651 34, 183-211. 652 Damesin, C., Rambal, S., Joffre, R., 1997. Between-tree variations in leaf delta13C of 653 Quercus pubescens and Quercus ilex among Mediterranean habitats with different 654 water availability. Oecologia 111, 26-35. 655 Díaz Barradas, M.C., Zunzunegui M., Ain-Lhout F.Z., Tirado R., García Novo. F., 1999 656 Plant functional types and ecosystem function in Mediterranean scrub. J. Veg. Sci. 657 10, 709-716. 658 Díaz, S., Hodgson, J.G., Thompson, K., Cabido, M., Cornelissen, J.H.C., Jalili, A., 659 Montserrat-Martí G., Grime J.P. et al., 2004. The plant traits that drive 660 ecosystems: Evidence from three continents. J. Veg. Sci. 15, 295–304. 661 Dietrich, W. and Wagner, W.L., 1988. Systematics of Oenothera section Oenothera 662 subsection Raimannia and subsection Nutantigemma (Onagraceae).Systematic 663 Botany Monographs 24. American Society of Plant Taxonomists, 91 pp. 664 Dietrich, W., 2000. Oenothera In: Castroviejo S. et al. (eds). Flora Iberica, vol. VIII: 665 Haloragaceae-euphorbiaceae. Real Jardín Botánico. CSIC. Madrid, pp. 90-100. 666 Dufour-Dror, J.M., 2013. Alien Invasive Plants in Israel. The Middle East Nature 667 Conservation Promotion Association. Ahva. Jerusalem. 668 Ehrenfeld, J.G., 2010. Ecosystem Consequences of Biological Invasions. Annual Rev. 669 Ecol. Evol. S. 41, 59-80. 670 Ehrendorfer, F., Guo, Y.P., 2005. Changes in the circumscription of the genus Achillea 671 (Compositae-Anthemidae) and its subdivision. Willdenowia 35, 49-54. 672 Flexas, J., Díaz-Espejo, A., Gago, J., Gallé, A., Galmés, J., Gulías, J., Medrano, H., 2014. 673 Photosynthetic limitations in Mediterranean plants: A review. Environ. Exp. Bot. 674 103, 12-23. 675 Flores-Moreno, H., Moles, A.T., 2013. A comparison of the recruitment success of 676 introduced and native species under natural conditions. PloS one, 8 (8), [e72509]. 677 Gallego, J., Martinez, M.L., García-Franco, J.G., Zunzunegui, M., 2019. The impact on 678 plant communities of an invasive alien herb, Oenothera drummondii, varies along 679 the beach-coastal dunes gradient. Flora. Doi: 680 doi.org/10.1016/j.flora.2019.151466. 681 24 García-de-Lomas, J., Fernández-Carrillo, L., Saavedra, M.C., Mangas, L., Rodríguez, C., 682 Sánchez-Gullón, E., Martínez, E., 2015. Invasión de Oenothera drummondii 683 Hook (Onagraceae) en el Paraje Natural Marismas del Odiel (Huelva, S España). 684 Bases para la gestión de una invasión avanzada. Rev. Soc. Gad. Hist. Nat. 9, 41685 50. 686 García-de-Lomas, J., Fernández-Carrillo, L., Saavedra, C., Dana, E.D., Rodríguez, C., 687 Martínez, E., 2016. Feasibility of using glyphosate to control beach evening 688 primrose Oenothera drummondii in heavily invaded coastal dunes, Odiel 689 Marshes, Spain. Conservation Evidence 13, 72-78. 690 García Novo, F., Merino, J., 1993. Dry coastal ecosystems of southwest Spain. In: van 691 der Maarel E (ed) Ecosystem of the world 2A: Dry coastal ecosystems, Polar 692 Regions and Europe. Elsevier, Amsterdam, pp. 349-362. 693 Garnier, E., Shipley, B., Roumet, C., Laurent, G., 2001. A standardized protocol for the 694 determination of specific leaf area and leaf dry matter content. Funct. Ecol. 15, 695 688–695. 696 Genty, B., Briantais, J.M., Baker, N.R., 1989. The relationship between the quantum yield 697 of photosynthetic electron transport and quenching of chlorophyll fluorescence. 698 Biochim. Biophys. Acta 990, 87-92. 699 Godoy, O., Pires de Lemos-Filhob, J., Valladares, F., 2011. Invasive species can handle 700 higher leaf temperature under water stress than Mediterranean natives. Environ 701 Exp. Bot. 71, 207–214. 702 Grammatikopoulos, G., Manetas, Y., 1994. Direct absorption of water by hairy leaves of 703 Phlomis fruticosa and its contribution to drought avoidance. Can. J. Bot. 72, 704 1805–1811. 705 Gratani, L., Bombelli, A., 1999. Leaf anatomy, inclination, and gas exchange 706 relationships in evergreen sclerophyllous and drought semideciduous shrub 707 species. Photosynthetica 37, 573–585. 708 Gratani, L., 2014. Plant phenotypic plasticity in response to environmental factors. Adv. 709 Bot. 2014. Article ID 208747. 710 Gutschick, V.P., Wiegel, F.W., 1988. Optimizing the Canopy Photosynthetic Rate by 711 Patterns of Investment in Specific Leaf Mass. The Am. Nat. 132, 67-86. 712 25 Havaux, M., Kloppstech, K., 2001. The protective functions of carotenoid and flavonoid 713 pigments against excess visible radiation at chilling temperature investigated in 714 Arabidopsis npq and tt mutant. Planta 213, 953-966. 715 He, P.J., Mao, B., Shen, C.M., 2013. Cultivation of Chlorella vulgaris on wastewater 716 containing high levels of ammonia for biodiesel production. Bioresour. Technol. 717 129, 177–181. 718 Hesp, P.A., 1991. Ecological processes and plant adaptations on coastal dunes. J. Arid 719 Environ. 21, 165-191. 720 Heyligers, P.C., 2008. Flora of the Stockton and Port Hunter sandy foreshores with 721 comments on fifteen notable introduced species. Cunninghamia 10, 493-511. 722 Hodgson, J.G., Montserrat-Marti, G., Charles, M., Jones, G., Wilsonm, P., Shipley, B., 723 Sharafi, M., Cerabolini, B.E.L., Cornelissen, J.H.C., Band, S.R., Bogard A., 724 Castro-Díez, P, Guerrero-Campo J, Palmer C, Pérez-Rontomé MC, Carter G, 725 Hynd A., Romo-Díez, A., de Torres-Espuny, L., Royo Pla, F., 2011. Is leaf dry 726 matter content a better predictor of soil fertility than specific leaf area? Ann Bot. 727 London 108, 1337–1345. 728 Hoffmann, A.A., Parsons, P.A., 1997. Extreme Environmental Change and Evolution. 729 Cambridge University Press, Cambridge (UK). 730 van Kleunen, M., Weber, E., Fischer, M., 2010. A meta-analysis of trait differences 731 between invasive and non-invasive plant species. Ecol. Lett. 13, 235-245. 732 Körner, C., Diemer, M., 1987. In situ photosynthetic responses to light, temperature and 733 carbon dioxide in herbaceous plants from low and high altitude. Funct. Ecol. 1, 734 179–194. 735 Liao, H., D'Antonio, C. M., Chen, B., Huang, Q., Peng, S., 2016. How much do 736 phenotypic plasticity and local genetic variation contribute to phenotypic 737 divergences along environmental gradients in widespread invasive plants? A 738 meta-analysis. Oikos 125: 905-917. 739 Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic 740 biomembranes. Methods in Enzymology, 148, 350-382. 741 Lichtenthaler, H.K., Buschmann, C., 2001. Chlorophylls and carotenoids: Measurement 742 and characterization by UV–VIS spectroscopy. Current Protocols in Food 743 Analytical Chemistry, John Wiley and Sons, New York (2001), pp. F4.3.1-F4.3.8. 744