Hydrobiologia https://doi.org/10.1007/s10750-018-3576-1 INVASIVE SPECIES II Regeneration and colonization abilities of the invasive species Elodea canadensis and Elodea nuttallii under a salt gradient: implications for freshwater invasibility Lise Thouvenot . Gabrielle Thiébaut Received: 6 June 2017 / Revised: 18 February 2018 / Accepted: 2 March 2018 Ó Springer International Publishing AG, part of Springer Nature 2018 Abstract Salinity plays an important role in macrophyte distribution. The current increase in salinization of native freshwaters could modify their susceptibility to invasion. In this study, we determined the tolerance to salt of two invasive macrophytes: Elodea canadensis and Elodea nuttallii. We analysed their growth, regeneration and colonization abilities and the influence of their phenological stage in their response to salt in a laboratory experiment. Traits of both Elodea species varied according to the season and the salt concentration: they were more affected by salt in autumn than in spring, demonstrating higher salt tolerance in spring than in autumn. The two species were sensitive to different thresholds of salinity, although they were both strongly reduced at 3 g l-1 of salt in autumn. Consequently, salt marshes and brackish waters (salt concentration inferior to 3 g l-1 of salt) are likely to be invaded by both species, but the Guest editors: John E. Havel, Sidinei M. Thomaz, Lee B. Kats, Katya E. Kovalenko & Luciano N. Santos / Aquatic Invasive Species II L. Thouvenot (&) German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Leipzig, Germany e-mail: lise.thouvenot@idiv.de G. Thiébaut Univ Rennes, CNRS, ECOBIO UMR 6553, 35000 Rennes, France effect of salt levels superior to 3 g l-1 on plant invasiveness needs to be investigated. Keywords Biological invasion
Invasiveness
Aquatic plants
Salinity
Traits Introduction In the last century, the number of introduced species in new areas has increased with the rise in international trade, despite efforts to limit invasions (Seebens et al., 2016). Non-native species of freshwater ecosystems are introduced accidentally through transport by boats (water ballast, attachment on boats), but more often intentionally for their ornamental qualities in the aquarium or water garden trade or for hobbies (Thiébaut, 2017b; Vila & Garcia-Berthou, 2010). Once released into non-native ecosystems, the establishment success of non-native species depends among others on the characteristics of the new habitat (Lonsdale, 1999). Abiotic and biotic factors affect the establishment of non-native species and are subject to numerous hypotheses relating to positive or negative effects on invasion success (e.g. ‘‘the fluctuating resource availability’’, Davis et al. (2000); or ‘‘Biotic resistance ‘‘, Elton (1958)). Disturbances, such as eutrophication or salinization, are thus key factors for assessing non-native species establishment success. For example, Chytry et al. (2008) have reported that 123 Hydrobiologia the most invasible habitats are rich in nutrients, whereas saline grasslands are resistant to invasion. The global increase of the salinization of freshwaters (due to agriculture, resource extraction and industrialization) and its impacts have been highlighted in several recent articles (Herbert et al., 2015; Cañedo-Argüelles et al., 2016). The concentration of 3 g l-1 is generally considered as the lower limit for saline waters (Hart et al., 1991): freshwaters are generally defined as water in which salt concentration is less than 3 g l-1 and marine water as water with a salt concentration of 35 g l-1. In addition, the salt concentration of freshwater is also reported to fluctuate intra-annually. In fact, freshwater salinity can undergo large seasonal fluctuations due to the concentration of salt in ground and surface waters during summer droughts, and to the runoff of salts in de-icing compounds during the winter season. Salinization of freshwaters and its variation could have serious consequences for ecosystem processes. As salt concentration increases, the species richness, abundance and growth of freshwater biota decrease (Hart et al., 1991; James & Hart, 1993; Brock et al., 2005; James et al., 2009). Salinity affects the physiology (Rout & Shaw, 2001; Tripathi et al., 2007; Abraham, 2010), the productivity (Haller et al., 1974; Twilley & Barko, 1990) and the distribution of plants, as well as the composition and structure of plant communities (Hart et al., 1991; Bertness et al., 1992; Sim et al., 2006; Smith et al., 2009; Goodman et al., 2010). In general, freshwater biota do not extend into saline or slightly saline water, and most freshwater plants do not tolerate increasing salt concentration (Nielsen et al., 2003). Consequently, salt could limit or enhance the abundance, distribution and expansion ranges of invasive macrophyte species in the subsequent decades. However, salt stress tolerance varies between plant species (Munns & Tester, 2008), and thus, the success of the invasion into inland waters will depend on the functional traits of macrophyte species (i.e. tolerance vs resistance to salt) and on the habitat characteristics (salt concentrations and fluctuations). In addition to high concentrations, fluctuations in salt concentrations and their timing (i.e. the season or the phenological stage of the plant when the stress occurs) may affect aquatic organisms, due to difficulties in regulating their osmotic balance (Munns, 2002; de Oliveira et al., 2013), and due to different sensitivities over their growth cycle. For example, Zedler et al. 123 (1990) showed that salt tolerances of Juncus kraussii Hochst. and Typha orientalis Presl. vary according to their phenological stage. Here, we focused our research on two invasive submerged macrophyte species native from North America: Elodea canadensis (Michaux) and Elodea nuttallii (Planchon) H. St John (Hydrocharitaceae). Both species have invaded numerous freshwaters in Europe, Asia and Australia (Cook & Urmi-König, 1985). Only female individuals were introduced into Europe, and the two species reproduce asexually by vegetative clonal propagation in their introduced area (Cook & Urmi-König, 1985). These rooted and perennial aquatic species have become dominant in invaded ecosystems and can form dense monospecific mats due to their abilities to tolerate a broad range of environmental conditions and different trophic levels (Cook & Urmi-König, 1985; Zehnsdorf et al., 2015). Hutchinson (1975) stated that E. canadensis grows well in salt level up to 0.5 g l-1, but later, other authors established that this species reduces its photosynthesis at low salt levels such as 0.1 g l-1 of chloride (Zimmermann-Timm, 2007) and that its biomass production was reduced to around 50% at 3 g l-1 of salt (Thouvenot et al., 2015). The salt tolerance of E. nuttallii is unknown. However, Hauenstein & Ramirez (1986) showed that the species E. densa, which is phylogenetically close to E. canadensis and E. nuttallii, is able to tolerate salt levels of up to 8 g l-1 in laboratory conditions. The aim of our study was to investigate the susceptibility to invasion by E. canadensis and E. nuttallii of aquatic ecosystems exposed to a salt gradient, using laboratory experiments. As salt concentrations of freshwater ecosystems increase simultaneously as a result of human activities and seasonal changes, we expect that this modification could greatly limit the invasion success of these two invasive species. We compared the functional responses of Elodea species to salt by analysing their morphological traits. Although the vegetative reproduction of Elodea species has been investigated already (BarratSegretain, 1996; Barrat-Segretain et al., 1998; BarratSegretain & Bornette, 2000), the effect of salinity on the invasive success of their vegetative propagules has been scarcely explored. We expected that the growth, colonization and regeneration abilities of propagules of both Elodea species would decrease as salt concentration increases. Furthermore, we Hydrobiologia hypothesized that the effect of the salt concentration on plant traits would vary according to the season. Materials and methods The laboratory experiment was carried out twice: the first in spring and the second in autumn. Two Elodea species were collected in spring and in autumn from the ‘‘Moder’’ river, in the northern Vosges (north-east of France (48°530 4200 N; 7°180 5100 E). The Moder River has fast-moving eutrophic water and drains a forested area, and this hydrological network flows through alluvial deposits poor in calcium carbonate. It is heavily colonized by macrophytes from early spring to late autumn. In the Moder River, E. canadensis has been observed since the end of the nineteenth century, and E. nuttallii was first recorded during the 1970s (Thiébaut, 2007a). All the shoots were sampled in a single plant species patch, and came from the same clonal individual for each species. Green shoots with apices of E. canadensis and E. nuttallii were selected. After 1 week of acclimation in tap water at ambient temperature, the two species were cleaned gently by hand to remove algae before being used for the experiment. Shoots of E. canadensis and E. nuttallii were cut into 10 cm lengths (mean ± SD: 9.97 ± 0.01 cm) after field collection in spring and in autumn. The selected shoot with its apex had no roots, no trace of necrosis, and no buds or lateral stems. Three individuals of E. canadensis or E. nuttallii were distributed randomly in a container [dimensions (L 9 W 9 H): 8 cm 9 8 cm 9 15 cm] which was filled with 750 ml of saline solution alone. We added no sediment to avoid salt adsorption by the sediment and because these plants can take up nutrients from the water (Thiébaut & Muller, 2003). The two species were exposed to five salt concentrations: 0; 0.3; 0.5; 1 and 3 g l-1, which corresponded to concentrations found in coastal marshes or inland rivers subject to industrial salt discharges (Piscart et al., 2006). Salt solutions were prepared by dissolving Red Sea salt (Red Sea Pharmaceuticals, Haifa, Israel) in dechlorinated tap water. Red Sea salt provides a widely relevant ionic proportion similar to seawater, which is predominantly sodium chloride, but in which calcium is also relatively important (Calcium: 400 ppm; magnesium: 1,300 ppm, potassium: 400 ppm, sodium: 400 ppm). The composition of tap water was conductivity = 635 lS.cm-1; pH = [NNO2-]7.7; [NNO3-] = 21.07 mg l-1; ? -1 \ 0.01 mg l ; [NNH4 ] B 0.02 mg l-1; -1 3[PPOH4 ] B 0.02 mg l . Four (in spring) or five (in autumn) replicates were used for each saline solution and species. All containers were placed randomly in a refrigerated tank with a temperature of 10°C, at a Photosynthetic Photon Flux Density (PPFD) of 21.45 lmol m-2 s-1 with a 12-h light/ 12-h dark cycle. This temperature was the mean temperature observed in spring and autumn in the east of France, and avoided algal growth. The water level in each container was maintained by adding dechlorinated tap water in order to offset losses from evaporation and to avoid increasing salt concentrations and plant desiccation. Six morphological traits on each plant were measured after 30 days of exposure to saline solution in the laboratory. The total stem length and Leaf Area (LA) were measured at the end of the experiment in order to evaluate the growth of plants according to a salinity gradient. The LA corresponded to the surface of one leaf taken at 3 cm on each plant apex: leaves were scanned and then measured using Scion Image software. We also measured the number and mean length of lateral shoots (i.e. regeneration ability), as well as the number and mean length of roots (i.e. colonization ability). Statistical analysis Statistical analyses were carried out using statistical RTM 2.13.0 software, with a significant threshold a = 0.05. We compared the abilities of each plant species to grow, regenerate and colonize freshwater habitats under different salt levels and depending on the season, with a three way ANCOVA (with species and seasons as factors and salt concentrations as the covariate). Although we did not use data transformation for the total shoot length and Leaf Area, we performed square root transformations for the number of lateral shoots, the number and length of roots and a logarithm transformation for the length of lateral shoots. When the ‘‘season’’ factor alone or interacting with species and/or salt concentration was significant (see ‘‘Results’’ section, Table 1), a simplification model was performed. Thus, the effect of species (as the factor) and salt concentration (as the covariate) 123 123 Table 1 Summary of the three-factor, ANCOVA with species and season as factors, and salinity concentration as a covariate, on the different morphological traits measured on Elodea canadensis and E. nuttallii in the two laboratory experiments Df N Species 1 135 Season 1 120–150 Stem length Leaf Area Number of lateral shoots Length of lateral shoots Number of roots Length of roots F P F P F F F F 19.29 < 0.0001 56.00 < 0.0001 71.96 < 0.0001 1.59 0.21 111.57 < 0.0001 93.03 < 0.0001 116.75 < 0.0001 4.99 0.03 P P 0.012 P P 0.91 2.16 0.14 53.50 < 0.0001 23.50 < 0.0001 < 0.0001 4.24 0.04 0.03 0.87 10.77 0.001 14.92 < 0.001 15.07 < 0.001 17.66 34.18 < 0.0001 5.43 0.02 2.94 0.09 3.14 0.08 14.77 < 0.001 0.14 0.71 7.83 < 0.001 6.89 0.01 1.26 0.26 0.10 0.75 12.35 < 0.001 5.52 0.02 24–30 25.17 < 0.0001 0.06 0.80 12.66 < 0.001 5.45 0.02 23.70 < 0.0001 16.11 < 0.0001 12–15 19.71 < 0.0001 0.07 0.78 6.82 0.01 0.39 0.53 0.15 0.70 4.15 0.04 Salinity 1 54 Species 9 season 1 60–75 Species 9 salinity 1 27 Season 9 salinity 1 Species 9 season 9 salinity 1 Significant results are in bold type Hydrobiologia Hydrobiologia was tested per season on each morphological trait of species with a two-way ANCOVA in order to observe the difference of traits along a salt gradient. To conduct the two-way ANCOVA, we performed square root transformations for the length of lateral shoot and number of roots in autumn and the number of lateral shoots in spring and autumn, as well as a logarithm transformation for the length of lateral shoots and the number of roots in spring. The adequate normality of the distribution of residuals was verified for each variable by checking the model plots and performing Shapiro–Wilk tests. Tukey’s HSD tests were applied to observe differences between treatments. Untransformed means and standard errors were used in the figures to facilitate interpretation. Results The response of macrophyte species to salt concentration depended on the three factors studied: species identity, season and salt concentration, both alone or in interaction. The stem length, Leaf Area as well as the production of lateral shoots differed between the two Elodea species, while the production and elongation of lateral shoots and roots varied according to the salt gradient (Table 1). However, season was often the most significant factor and affected all plant traits (Table 1). Indeed, growth (i.e. stem length and LA), regeneration (i.e. the production and elongation of lateral shoots), and colonization (i.e. the production and elongation of roots) possessed lower values of traits in spring than in autumn. In spring, salt concentration mainly affected the stem length, as well as root production and elongation. We observed that the stem length of E. nutallii was stimulated at the highest concentrations of 1 and 3 g l-1 of salt, while the stem length of E. canadensis was similar along the salt gradient (Df = 1, F = 17.26, P \ 0.0001, Fig. 1a). Although the Leaf Area was significantly lower for E. canadensis than for E. nuttallii (Df = 1, F = 2.41, P \ 0.0001, Fig. 1b), and that E. canadensis produced more lateral shoots than E. nuttallii (Df = 1, F = 30.89, P \ 0.0001, Fig. 1c), these two traits and the lateral shoots elongation (Fig. 1d) were not affected by salt concentrations in spring, in contrast to the root production. Indeed, the production of roots by E. canadensis at the 0.5 g l-1 concentration and by E. nuttallii at the 1 g l-1 concentration were higher than those of E. nuttallii at the 0 and 0.5 g l-1 concentrations of salt (Df = 1, F = 5.95, P = 0.02, Fig. 1e). The 1 and 3 g l-1 concentrations of salt stimulated the elongation of roots of E. nuttallii, whereas these same concentrations did not affect the elongation of roots produced by E. canadensis. Furthermore, the elongation of roots of E. nuttallii was higher at 1 g l-1 of salt, in contrast to the length of roots produced by E. canadensis at 3 g l-1 of salt (Df = 1, F = 13.25, P \ 0.001, Fig. 1f). These trait responses were different from those observed in autumn. At the highest salt concentration (3 g l-1 of salt), the stem length of both species was reduced (Df = 1, F = 42.37, P \ 0.0001, Fig. 2a), but E. nuttallii had a higher stem length in autumn than E. canadensis (Df = 1, F = 91.50, P \ 0.0001). Furthermore, the Leaf Area depended on the interaction between salt concentration and species identity (Df = 1, F = 4.09, P = 0.04, Fig. 2b): we observed a higher value of E. nuttallii LA at the 0.3 g l-1 of salt than at 1 g l-1, while E. canadensis LA was lower at 0.3 g l-1 than at 1 g l-1 of salt. The regeneration ability decreased drastically at 3 g l-1 of salt for E. canadensis (Fig. 2c, d), whereas only the length of lateral shoots of both species was reduced from salinities of 0.5 g l-1 of salt (Df = 1, F = 21.41, P \ 0.0001, Fig. 2d). The root production of both Elodea sp. decreased at 3 g l-1 of salt, but this decrease was significantly higher for E. canadensis species than for E. nuttallii (Df = 1, F = 9.25, P = 0.003, Fig. 2e). Similarly, the length of roots of both species decreased at 3 g l-1 of salt (Df = 1, F = 26.30, P \ 0.0001, Fig. 2f). Discussion The main result of our research was that the salt concentration of freshwaters could limit the development and persistence of the congeneric species E. canadensis and E. nuttallii, depending on the season. Indeed, our results showed that plant growth, regeneration and colonization abilities decreased as salt concentration increased, thus confirming our first hypothesis. Higher salinity levels had significant inhibitory effects on both Elodea species. When exposed to salt, plant survival, growth, productivity, photosynthesis and development are reduced (Hart et al., 1991; Parida & Das, 2005; McGregor et al., 123 Hydrobiologia (b) 0.30 25 Stem length (cm) in spring Species x Salinity : F=17.26; p < 0.0001 a 20 ab ab 15 b b 10 5 bc bcd bcd cd Leaf Area (cm²) in spring (a) 0.15 0.10 0.05 Species : F=2.41; p < 0.001 0.00 0 g.L-1 0.3 g.L-1 0.5 g.L-1 1g.L -1 3 g.L-1 0 g.L-1 (d) 3.0 Species : F=30.89; p < 0.0001 Mean length of lateral shoots (cm) in spring Mean number of lateral shoots in spring 0.20 d 0 (c) 0.25 2.5 2.0 1.5 1.0 0.5 0.0 Mean number of roots in spring (e) 3.5 3.0 0.3 g.L-1 0.5 g.L-1 1g.L -1 a 2.5 a 2.0 ab 1.5 ab ab ab 1.0 0.5 bc abc abc c 0.0 1g.L 0.5 g.L-1 1g.L -1 3 g.L-1 7 6 Ns 5 4 3 2 1 0 g.L-1 (f) Species : F=7.50; p = 0.007 Species x Salinity : F=5.95; p = 0.02 0.5 g.L-1 0 3 g.L-1 Mean length of roots (cm) in spring 0 g.L-1 0.3 g.L-1 0.3 g.L-1 -1 3 g.L-1 25 Species x Salinity : F=13.25; p <0.0001 20 15 ab ab abc bc abc 10 abc 5 a ab bc c 0 0 g.L-1 0.3 g.L-1 0.5 g.L-1 1g.L -1 3 g.L-1 0 g.L-1 0.3 g.L-1 0.5 g.L-1 1g.L -1 3 g.L-1 Fig. 1 Morphological traits (mean ± SE) of E. canadensis (white circles) and E. nuttallii (black circles) in spring along the salt gradient, after a 30-day experiment in the laboratory (a stem length, b Leaf Area, c number of lateral shoots, d length of lateral shoots, e number of roots and f length of roots). Results were obtained from the two-factor ANCOVAs (with species as the factor and salt concentrations as a covariate), on the different morphological traits measured for both species E. canadensis and E. nuttallii in spring (salt: Df = 1; n = 24; species: Df = 1; n = 60; species 9 salt: Df = 1; n = 12). Different small letters indicate significant differences between the interaction of species and salinity concentrations 2007; Munns & Tester, 2008). Petjukevics et al. (2015) showed that the long-term influence of salt in small concentrations activates the synthesis of carotenoids in Elodea canadensis, as a defensive reaction response to adverse environmental factors, whereas prolonged exposure to high concentrations of 123 a 25 20 b 15 10 5 Species : F=91.50; p <0.0001 Salinity : F=42.37; p <0.0001 Mean number of lateral shoots in autumn 0.3 g.L-1 0.5 g.L-1 1g.L -1 ab ab ab bc bc bc c 1.0 0.0 c c Species : F=41.83; p < 0.0001 Salinity : F=17.53; p < 0.0001 Species x Salinity : F=4.95; p = 0.03 0 g.L-1 0.3 g.L-1 0.5 g.L-1 Mean number of roots in autumn ab b 0.05 b bc bc 0.10 bc c Species : F=45.52; p <0.0001 Species x Salinity : F=4.09; p =0.04 0 g.L-1 1g.L 3 6 3.0 a 2.5 ab ab ab 2.0 ab 1.5 b ab ab 1.0 Species : F=9.75; p = 0.003 Salinity : F=58.45; p < 0.0001 Species x Salinity : F=9.25; p = 0.003 0 g.L-1 0.3 g.L-1 0.5 g.L-1 1g.L -1 c 0.5 g.L-1 1g.L -1 3 g.L-1 a ab 5 bc 4 bc c 3 2 1 Salinity : F= 21.41; p < 0.0001 0 g.L-1 g.L-1 (f) 0.3 g.L-1 7 0.3 g.L-1 0.5 g.L-1 1g.L -1 3 g.L-1 25 a 0.0 ab ab 0.15 0 -1 (e) 3.5 0.5 a 0.20 (d) 2.0 0.5 0.25 3 g.L-1 a 3.0 1.5 0.30 0.00 0 g.L-1 2.5 (b) Mean length of lateral shoots (cm) in autumn 0 (c) a a a Mean length of rrots (cm) in autumn Stem length (cm) in autumn (a) Leaf Area (cm²) in autumn Hydrobiologia a 20 ab 15 ab bc c 10 5 Salinity : F=26.30; p <0.0001 0 3 g.L-1 0 g.L-1 0.3 g.L-1 0.5 g.L-1 1g.L -1 3 g.L-1 Fig. 2 Morphological traits (mean ± SE) of E. canadensis (white circles) and E. nuttallii (black circles) in autumn along the salt gradient, after a 30-day experiment in the laboratory (a stem length, b Leaf Area, c number of lateral shoots, d length of lateral shoots, e number of roots and f length of roots). Results were obtained from the two-factor ANCOVAs (with species as the factor and salt concentrations as a covariate), on the different morphological traits measured for both species E. canadensis and E. nuttallii in autumn (salt: Df = 1; n = 30; species: Df = 1; n = 75; species 9 salt: Df = 1; n = 15). Different small letters indicate significant differences between salinity concentrations, or the difference between the interactions of species and salinity concentrations salt disturbed the physiological processes in plants and blocked processes of pigment synthesis. In addition, although niche overlaps could occur between both species due to their high degree of ecological and functional redundancy (Herault et al., 2008; Zehnsdorf et al., 2015), we detected different salinity tolerance 123 Hydrobiologia thresholds between them. We observed that the growth of E. canadensis decreased from a specific threshold of 1 g l-1 of salt, while the growth and root length of E. nuttallii were stimulated in spring at the higher concentrations of 1 and 3 g l-1, contrary to the lowest salinity levels in spring, when E. nuttallii seemed to lose biomass. Although stimulation of apical growth and reproduction was also observed in Schoenoplectus triqueter (L.) at low salinity concentrations (Deegan et al., 2005), most salt treatments induced biomass reduction. Biomass reduction could be due to the loss of leaves induced by premature leaf senescence or could be related to leaf cell expansion (van den Brink & van der Velde, 1993; Warwick & Bailey, 1997; Warwick & Bailey, 1998) which would correspond to a reduction of Leaf Area, attributed to a decrease in leaf gain and greater leaf loss, particularly at high salinity (e.g. Leaf Area of E. nuttalli at 1 g l-1 in autumn). Similar responses have been found for other aquatic macrophytes exposed to saline conditions (Haller et al., 1974; Rout & Shaw, 2001; Jampeetong & Brix, 2009; Thouvenot et al., 2015). Furthermore, the growth decrease could also be due to the compartmentalization and accumulation of inorganic ions in old plant parts, which are then sacrificed. This mechanism, allowing salt limitation in the youngest tissues, was described in glycophyte plants (Cheeseman, 1988). Another explanation could be the decrease of turgor induced by stress, causing the suppression or inactivation of elongation, thus allowing the plant to preserve limited energy in order to allocate its energy to defences and to limit the risk of heritable damage (May et al., 1998; Blindow et al., 2003; Lokhande et al., 2010). Here, it appeared that the high concentration of salt (3 g l-1) in spring could stimulate the development of E. nuttallii but inhibited the growth of E. canadensis, while at this same concentration in autumn, their growth decreased and their development was inhibited. The ramification pattern observed, i.e. the decrease of lateral shoot production and elongation, could be due to the same mechanism observed for the decrease of apical growth (i.e. decrease of turgor) leading to the preservation of energy, and the allocation energy to defences. Here, we observed that the regenerations of E. canadensis and E. nuttallii were globally higher in autumn, but that season and salinity affected them differently, suggesting their different abilities to regenerate in habitats with elevated levels of salt. 123 Regeneration and colonization would not be affected by salinity elevation in spring for either species and consequently may not be stopped. However, in autumn, regeneration would be inhibited for both species at 0.5 and 3 g l-1 of salt, respectively, leading to a decrease of the colonization abilities of the two plant species. Similarly, the production of roots and their length, simultaneously allowing the anchorage and establishment of plant species (i.e. colonization) and resource acquisition, were reduced at 3 g l-1 of salt. These findings are congruent with data in the literature. Freshwater macrophytes reduce root development from salinities of 1 g l-1 (Nielsen et al., 2003), which could be a mechanism to control the ion uptake in a stressful environment and is probably caused by the reduction in turgor pressure (Jampeetong & Brix, 2009). Reduction of root production could also be interpreted as a mechanism to limit anchorage into sediment to favour dispersal from areas with high salinity towards a less stressful environment. Thus, production and elongation of roots of the both species were limited with salinity elevation at a similar threshold (i.e. of 3 g l-1 of salt for root production and of 0.3 g l-1 of salt for root elongation), which would limit resource acquisition and plant establishment in new habitats. Furthermore, as hypothesized, we found that all morphological traits for both species varied according to the season, suggesting that the response to salt stress changed seasonally and that the sensitivity of plants to salt depends on the different stages of growth (Zedler et al., 1990). According to Di Nino et al. (2007) and Thiébaut et al. (2008), in natural environments, apical and lateral growths of both Elodea species begin at the end of winter, by using reserves in their dormant apices or turions, while at the end of summer, apical and lateral growths stop (Di Nino et al., 2007; Thiébaut et al., 2008). Turions (i.e. winter buds) of Elodea species were formed in autumn and were able to resist unfavourable conditions (Barrat-Segretain, 1996) by staying alive in the sediment during winter. This allowed the species to reproduce earlier in the spring by using carbohydrate, starch and nutrient (N and P) reserves stored in turions for growth during this period (Adamec, 2010; Xie et al., 2014), and thus the young shoots could allocate their energy to apical growth. In our study, a small apical growth of plants unexposed to stress was observed in spring. The harsh and long winter could explain the delay in plant Hydrobiologia development. In autumn, plants mobilized the accumulated internal resources in stems and leaves during their vegetative growth periods (spring and summer) in order to continue their development (i.e. the higher production levels of lateral shoots and roots were in autumn versus spring), to produce turions and/or to fight against different environmental constraints. Thus, the life cycle of both Elodea species could explain the differences between trait values measured in spring and in autumn. More surprisingly, a lower impact of salinity on the development of both species was measured in spring than in autumn. Resource accumulation in plants can affect the stress tolerance of plants. For example, young plants of Schoenoplectus triqueter were less tolerant to salinity than older plants (Deegan et al., 2005), and adult plants could compensate for salt stress with their stored reserves (French & Moore, 2003). Thus, nutrient storage in plant tissue at the end of winter could reduce the toxicity of the salt for Elodea species in spring, whereas in autumn, the species came close to depleting their resources and became more sensitive to salinity. Thus, our study showed that a salinity threshold of 3 g l-1 decreased the growth, the regeneration and colonization abilities for both species in autumn, but did not stop them. However, this salt concentration stimulated E. nuttallii growth and root length in spring. This species seems more tolerant to salinity than E. canadensis. Our results must also be observed with caution; the laboratory conditions used did not take into account several environmental factors which would be acting in the field (i.e. water temperature, velocity, nutrient content or biotic interactions, as well as salinity composition and pulses). Consequently, it is difficult to determine the exact salinity level that is toxic to a plant, due to the interactive effect of all of these environmental factors with salinity (CañedoArgüelles et al., 2013). Furthermore, we demonstrated that tolerances to salinity varied between congeneric species and between seasons. These findings show that eradication plans and management of invasive species should be species-specific. Therefore, it is important for land managers to identify the potential distribution of both Elodea species in time. 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