Differential Effects of Increasing Salinity on Germination and Seedling Growth of Native and Exotic Invasive Cordgrasses

plants Article Differential Effects of Increasing Salinity on Germination and Seedling Growth of Native and Exotic Invasive Cordgrasses María Dolores Infante-Izquierdo 1,*, Jesús M. Castillo 2, Brenda J. Grewell 3, F. Javier J. Nieva 1and Adolfo F. Muñoz-Rodríguez 1 1 Departamento de Ciencias Integradas. Fuerzas Armadas Ave., Campus El Carmen, Universidad de Huelva, 21071 Huelva, Spain; [email protected] (F.J.J.N.); [email protected] (A.F.M-R.) 2Departamento de Biología Vegetal y Ecología. Universidad de Sevilla, Ap. 1095, 41080 Sevilla, Spain; [email protected] 3USDA-ARS Invasive Species and Pollinator Health Research Unit, Department of Plant Sciences MS-4, 1 Shields Ave., University of California, Davis, CA 95616, USA; [email protected] *Correspondence: mariadolor[email protected] Received: 16 August 2019; Accepted: 23 September 2019; Published: 25 September 2019   Abstract: Soil salinity is a key environmental factor influencing germination and seedling establishment in salt marshes. Global warming and sea level rise are changing estuarine salinity, and may modify the colonization ability of halophytes. We evaluated the effects of increasing salinity on germination and seedling growth of native Spartina maritima and invasive S. densiflora from wetlands of the Odiel-Tinto Estuary. Responses were assessed following salinity exposure from fresh water to hypersaline conditions and germination recovery of non-germinated seeds when transferred to fresh water. The germination of both species was inhibited and delayed at high salinities, while pre-exposure to salinity accelerated the speed of germination in recovery assays compared to non-pre-exposed seeds. S. densiflora was more tolerant of salinity at germination than S. maritima. S. densiflora was able to germinate at hypersalinity and its germination percentage decreased at higher salinities compared to S. maritima. In contrast, S. maritima showed higher salinity tolerance in relation to seedling growth. Contrasting results were observed with differences in the tidal elevation of populations. Our results suggest S. maritima is a specialist species with respect to salinity, while S. densiflora is a generalist capable of germination of growth under suboptimal conditions. Invasive S. densiflora has greater capacity than native S. maritima to establish from seed with continued climate change and sea level rise. Keywords: climate change; dormancy; Odiel Marshes; quiescent seed; salinity tolerance; sea level rise; radicle 1. Introduction Salt marshes are highly stressful environments where halophytes are subjected to high mortality risk [ 1 ]. In these habitats, soil salinity is one of the key environmental factors determining vegetation distribution, partially by limiting seed germination and seedling establishment [ 2 ]. These phases are crucial in the life cycle of halophytes [ 3 – 5 ]. The general behaviour of halophytic seeds in the presence of salt is well documented [ 6 ]. Seeds of most halophytes show optimal germination in freshwater, differing in their germination responses to higher salinities [ 6 – 8 ]. High salinities usually inhibit germination of halophytes, however, some seeds maintain viability and are able to germinate when osmotic stress decreases [9–11]. Plants 2019,8, 372; doi:10.3390/plants8100372 www.mdpi.com/journal/plants Plants 2019,8, 372 2 of 15 Estuarine salt marshes are increasingly impacted by biological invasions [ 12 ]. Some invasive halophytes show high tolerance to salinity during germination and seedling growth and have colonized a wide range of salt-affected habitats [ 13 – 15 ]. In tidal wetlands, climate change and associated sea level rise are changing estuarine salinity patterns [ 16 ]. Local environmental conditions can be highly variable with climate change. Salinity decreases in some salt marshes due to an increase in rainfall, while salinity increases in other locations due to sea level rise and increases in temperature and evapotranspiration rates [ 17 – 19 ]. These environmental changes may modify the ability of native species to colonize new sites as well as the capacity of invasive halophytes to invade them. Cordgrasses (genus Spartina) provide a model halophyte group to study the responses of native and invasive species to environmental conditions since they inhabit salt marshes around the world, and many species have naturalized in habitats beyond their native ranges [ 20 ]. Specifically, native Spartina maritima (Curtis) Fernald and invasive Spartina densiflora Brongn. co-occur in salt marshes along the Gulf of C á diz (Southwest Iberian Peninsula) [ 21 ]. S. maritima is the only native cordgrass in European marshes [ 22 ], where it is a primary colonizer at low tidal elevations and facilitates the development of ecological succession [ 23 ]. Therefore, the conservation of this species is crucial for the maintenance of biodiversity in these ecosystems. The effects of salinity on seed germination and seedling growth have never been studied for native S. maritima. Actually, seed production of this species has been described as very low or non-existent [ 22 – 24 ], but we recently discovered that S. maritima in the Southwest Iberian Peninsula produces a moderate number of caryopses (13%) with high variation among tussocks (0%–45%), high viability (89%) and has high germination rates in freshwater (85%) [ 25 ]. In contrast, South American S. densiflora is one of the three most widely distributed species of the genus and was introduced to the Southwest Iberian Peninsula centuries ago [ 26 ]. S. densiflora shows high tolerance to environmental variation, including salinity levels [ 27 ]. This niche breath has resulted in its colonization of a wide range of different habitats along the intertidal gradient [ 21 , 26 , 28 ]. Seed production is key to the spread of Spartina species [ 20 ], and the ability of S. densiflora to germinate is a recognized determinant for its invasive potential from brackish marshes to hypersaline saltpans [ 29 – 31 ]. To our knowledge, no previous study has evaluated salinity responses of S. densiflora seeds that were produced in different habitats along the intertidal gradient. Previous studies of halophytes have found that germination tolerance to salinity depends on environmental conditions in the source habitats where they were produced [9,11,32]. Our main goals were to analyse germination and seedling growth of native S. maritima and invasive S. densiflora in response to salinity ranging from freshwater to hypersaline levels. We hypothesized that S. maritima seeds and seedlings would show high salinity tolerance since the species colonizes low elevation tidal marshes where medium–high salinity levels occur throughout the year [ 33 ]. We also hypothesized that S. densiflora would show high plasticity in response to salinity since it has invaded a wide range of habitats with contrasted salinity regimes [ 21 ]. Germination experiments evaluating the responses of these species to salinity ranging from freshwater to hypersalinity, and recovery of the species after salinity release, were carried out under controlled conditions to test our hypotheses. Results were compared to field conditions where propagules were sourced for the experiments. 2. Results 2.1. Germination Responses to Salinity Spartina maritima achieved its highest germination percentage (c. 96 %) at low salinity levels between 0.00 and 0.15 M NaCl. Germination rate for the native species decreased at higher salinities. No seed was able to germinate at 0.75 M NaCl. T 50 G (days necessary to reach 50% of the final germination percentage) was increased at salinities higher than 0.30 M NaCl (Table 1). Germination percentage for S. densiflora decreased significantly in salinities higher than 0.30 M NaCl in seeds from all locations and habitats (Figure 1). However, at higher salinities, the germination percentage of seeds from LM (low marsh) was more than double than those from MM (middle marsh) Plants 2019,8, 372 3 of 15 and HM (high marsh) (Figure 1). Seeds produced in LM showed higher germination (19% ± 3%) than those from MM and HM (c. 9%) at hypersalinity (0.75 M NaCl) (Kruskal–Wallis test, H 2,36 =9.16, p<0.05) (Figure 1a–c). Plants 2019, 8, 372 4 of 16 Figure 1. Percentages of germination (white bars), recovery of germination after salt exposure (grey bars) and seed dormancy (black bars) in six salinity treatments for Spartina densiflora from three habitats (grouping the three locations in each habitat): (a) low marsh (LM), (b) middle marsh (MM), (c) high marsh (HM), and from three locations (grouping the three habitats in each location): (d) Almendral, (e) Bacuta, (f) Calatilla, in the Odiel Marshes (Southwest Iberian Peninsula). Data are mean ± SE (n = 4). Different letters indicate significant differences among treatments for each trait (Mann–Whitney U test, in italic, for Kruskal–Wallis or Tukey’s HSD test, in non-italic, for one-way ANOVA, p < 0.05). Figure 1. Percentages of germination (white bars), recovery of germination after salt exposure (grey bars) and seed dormancy (black bars) in six salinity treatments for Spartina densiflora from three habitats (grouping the three locations in each habitat): ( a ) low marsh (LM), ( b ) middle marsh (MM), ( c ) high marsh (HM), and from three locations (grouping the three habitats in each location): ( d ) Almendral, ( e ) Bacuta, ( f ) Calatilla, in the Odiel Marshes (Southwest Iberian Peninsula). Data are mean ± SE (n =4). Different letters indicate significant differences among treatments for each trait (Mann–Whitney U test, in italic, for Kruskal–Wallis or Tukey’s HSD test, in non-italic, for one-way ANOVA, p<0.05). Plants 2019,8, 372 4 of 15 Table 1. Germination percentage (G), T 50 of germination (T 50 G), recovery germination percentage after salt exposure (RG), T 50 of germination recovery (T 50 R), and seed viability percentage (V) for native Spartina maritima from the Gulf of C á diz (Southwest Iberian Peninsula) in six salt treatments. Data show mean ± Standard Error (SE) (n =3–4). Different letters indicate significant differences between treatments (Mann–Whitney U test, in italic, for Kruskal–Wallis or Tukey’s Honest Significant Difference (HSD) test, in non-italic, for Analysis of Variance (one-way ANOVA), p<0.05). df (degrees of freedom). Salinity (M NaCl) G (%) T50 G (days) RG (%) T50 R (days) V (%) 0.00 100 ±0a 23 ±1a - - 76 ±6a 0.15 91 ±4a 28 ±2ab 9 ±4a 3 ±1a76 ±8a 0.30 51 ±6b 32 ±4abc 49 ±6b 6 ±1a76 ±7a 0.45 19 ±4c 38 ±6bc 81 ±4c 11 ±1b78 ±1a 0.60 12 ±5cd 43 ±3c 88 ±5cd 12 ±1b71 ±3a 0.75 0 ±0d - 100 ±0d 13 ±0b78 ±3a one-way ANOVA (F) or Kruskal–Wallis (H) test F=119.17, df =5, p<0.0001 F=5.43, df =4, p<0.01 F=74.59, df =4, p<0.0001 H4,19 =14.64, p<0.01 F=0.22, df =5, p>0.05 Increasing salinity decreased the germination speed (higher T 50 G) for seeds sourced from all marsh elevation zones and locations. Additionally, S. densiflora seeds produced at LM germinated 27% faster in freshwater than those from MM and HM (one-way ANOVA, F =11.03, degrees of freedom (df) =2, p<0.0005) (Table 2). Table 2. Comparisons among salt treatments for Spartina densiflora in the Southwest Iberian Peninsula. Percentage of viability (V), T 50 of germination (T 50 G) and T 50 of recovery (T 50 R) in the different salt treatments (0.00, 0.15, 0.30, 0.45, 0.60 and 0.75 M NaCl) for different habitats and locations. Data are mean ± SE (n =12). Different letters indicate significant differences among treatments for each trait (Mann–Whitney U test, in italic, for Kruskal–Wallis or Tukey’s HSD test, in non-italic, for one-way ANOVA, p<0.05). df (degrees of freedom). Salinity (M NaCl) V (%) T50 G (days) T50 R (Days) V (%) T50 G (days) T50 R (days) Low marsh ‘Almendral’ 0.00 79 ±3a 19 ±1a - 65 ±6a 22 ±1a - 0.15 82 ±3a 22 ±1ab 24 ±5a65 ±6a 25 ±1ab 20 ±4a 0.30 80 ±3a 25 ±2b 15 ±3ab 62 ±7a 27 ±2abd 12 ±3ab 0.45 79 ±4a 27 ±2bc 9 ±1bc 57 ±8a 32 ±3bc 8 ±1bc 0.60 73 ±4a 33 ±3c 6 ±1cd 55 ±7a 40 ±4c 5 ±1c 0.75 71 ±3a 36 ±2c 5 ±1d52 ±8a 39 ±4cd 6 ±0bc one-way ANOVA (F) or Kruskal–Wallis (H) test F=1.77, df =5 p>0.05 F=14.16, df =5 p<0.0001 H4,55 =17.51 p<0.005 F=0.61, df =5 p>0.05 F=9.38, df =5 p<0.0001 H4,53 =19.85 p<0.001 Middle marsh ‘Bacuta’ 0.00 70 ±4a 26 ±1a- 66 ±5a 23 ±1a- 0.15 64 ±2ab 28 ±2ab 11 ±2a63 ±5a 27 ±2ab 19 ±6a 0.30 56 ±2bc 31 ±2ab 9±3a57 ±6a 28 ±2b13 ±3a 0.45 45 ±3cd 37 ±4bc 12 ±4a44 ±6a 31 ±3b13 ±4a 0.60 41 ±3d 39 ±8abc 7±1a47 ±6a 31 ±5abc 7±1a 0.75 39 ±3d 43 ±5c7±0a46 ±5a 43 ±4c6±1a one-way ANOVA (F) or Kruskal–Wallis (H) test F=18.08, df =5 p<0.0001 H5,62 =13.71 p<0.05 H4,51 =5.08 p>0.05 F=2.91, df =5 p<0.05 H5,63 =20.42 p<0.005 H4,51 =6.27 p=0.180 High marsh ‘Calatilla’ 0.00 54 ±4a 25 ±1a- 71 ±3a 24 ±2a- 0.15 46 ±3ab 25 ±1a31 ±9a64 ±3a 24 ±2a25 ±8a 0.30 37 ±3bc 28 ±2ab 13 ±3ab 54 ±5ab 29 ±3ab 11 ±3a Plants 2019,8, 372 5 of 15 Table 2. Cont. Salinity (M NaCl) V (%) T50 G (days) T50 R (Days) V (%) T50 G (days) T50 R (days) 0.45 32 ±3c 36 ±4abc 7±1b55 ±5ab 38 ±5ab 7±1a 0.60 30 ±2c 37 ±5bc 6±1b43 ±5b 37 ±5b6±1a 0.75 30 ±3c 42 ±6c7±1b42 ±5b 33 ±1b6±1a one-way ANOVA (F) or Kruskal–Wallis (H) test F=10.14, df =5 p<0.0001 H5,57 =16.94 p<0.005 H4,49 =14.91 p<0.005 F=6.81, df =5 p<0.0001 H5,62 =12.99 p<0.05 H4,51 =6.21 p>0.05 2.2. Germination Responses after Salinity Exposure The germination rate of Spartina maritima seeds increased, and the speed of germination decreased significantly during recovery after exposure to higher salinities. T 50 R (days necessary to reach 50% of the final germination percentage in recovery assays) was reduced after recovery from all levels of salinity exposure than seeds that were germinated in freshwater. The T 50 reduction indicating an increase in germination speed was most extreme during recovery from 0.15 M NaCl exposure when germination accelerated almost nine times (Table 1). In contrast to S. densiflora, no dormant seeds were recorded for S. maritima during the recovery experiment. S. maritima seed viability was c. 76%, without showing significant differences among salinities (Table 1). As in the case of S. maritima,S. densiflora seeds had increased germination rates during recovery following exposure to higher salinities, and this result held for seeds sourced from every location and within-marsh habitat elevation zone (Figure 1). S. densiflora seeds tended to have increased germination speeds (lower T 50 R) during recovery following exposure to higher salinities (Table 2). Seeds from LM exposed previously to hypersalinity showed lower recovery germination percentage (78% ± 3%) and lower T 50 (5 ± 1 days) than seeds from MM and HM (c. 90% and 7 days, respectively) (Figure 1, Table 2). Dormant S. densiflora seeds in freshwater were c. 7% for every habitat and location and tended to decrease with increasing salinity exposure (Figure 1). Seed viability in control treatments were higher for seeds of S. densiflora from LM and MM (c. 76%) than those from HM (54% ± 4%) (one-way ANOVA, F =12.95, df =2, p<0.0001). On the other hand, seed viability was not affected by salt treatments in seeds from LM, while in seeds from MM and HM the viability decreased as salinity increased (Table 2). Regarding the three source population locations, there were no significant differences in seed viability at any salinity level for seed sourced from Almendral, whereas viability was reduced at higher salinities for the other two study locations (Table 2). 2.3. Initial Seedling Growth Responses to Salinity S. maritima seedlings had similar cotyledon and first leaf lengths at every salinity level (Figure 2). However, the radicles of S. maritima were four times longer at 0.15 M NaCl than radicles that emerged from seeds exposed to other salinity concentrations (Kruskal–Wallis test, H 3,59 =8.66, p<0.05). Cotyledon, first leaf and radicle of S. maritima seedlings growing in freshwater all increased in length in recovery after seeds had been exposed to salinity higher than 0.15 M NaCl (Figure 2). Plants 2019,8, 372 6 of 15 Plants 2019, 8, 372 6 of 16 dormant seeds were recorded for S. maritima during the recovery experiment. S. maritima seed viability was c. 76%, without showing significant differences among salinities (Table 1). As in the case of S. maritima, S. densiflora seeds had increased germination rates during recovery following exposure to higher salinities, and this result held for seeds sourced from every location and within-marsh habitat elevation zone (Figure 1). S. densiflora seeds tended to have increased germination speeds (lower T 50 R) during recovery following exposure to higher salinities (Table 2). Seeds from LM exposed previously to hypersalinity showed lower recovery germination percentage (78% ± 3%) and lower T 50 (5 ± 1 days) than seeds from MM and HM (c. 90% and 7 days, respectively) (Figure 1, Table 2). Dormant S. densiflora seeds in freshwater were c. 7% for every habitat and location and tended to decrease with increasing salinity exposure (Figure 1). Seed viability in control treatments were higher for seeds of S. densiflora from LM and MM (c. 76%) than those from HM (54% ± 4%) (one-way ANOVA, F = 12.95, df = 2, p < 0.0001). On the other hand, seed viability was not affected by salt treatments in seeds from LM, while in seeds from MM and HM the viability decreased as salinity increased (Table 2). Regarding the three source population locations, there were no significant differences in seed viability at any salinity level for seed sourced from Almendral, whereas viability was reduced at higher salinities for the other two study locations (Table 2). 2.3. Initial Seedling Growth Responses to Salinity S. maritima seedlings had similar cotyledon and first leaf lengths at every salinity level (Figure 2). However, the radicles of S. maritima were four times longer at 0.15 M NaCl than radicles that emerged from seeds exposed to other salinity concentrations (Kruskal–Wallis test, H 3,59 = 8.66, p < 0.05). Cotyledon, first leaf and radicle of S. maritima seedlings growing in freshwater all increased in length in recovery after seeds had been exposed to salinity higher than 0.15 M NaCl (Figure 2). Figure 2. Cotyledon (white bars), first leaf (grey bars) and radicle length (black bars) for seedlings of Spartina maritima germinated: (a) in six salt treatments and (b) in the recovery (R) assays after salinity exposure. Data show mean ± SE (n = 5–13). Different letters indicate significant differences between salinity treatments (Mann–Whitney U test, in italic, for Kruskal–Wallis or Tukey’s HSD test, in nonitalic, for one-way ANOVA, p < 0.05). Asterisks and plus sign indicate significant differences Figure 2. Cotyledon (white bars), first leaf (grey bars) and radicle length (black bars) for seedlings of Spartina maritima germinated: ( a ) in six salt treatments and ( b ) in the recovery (R) assays after salinity exposure. Data show mean ± SE (n =5–13). Different letters indicate significant differences between salinity treatments (Mann–Whitney U test, in italic, for Kruskal–Wallis or Tukey’s HSD test, in non-italic, for one-way ANOVA, p<0.05). Asterisks and plus sign indicate significant differences compared to control treatment (0.00 M) for each seedling parameter (Mann–Whitney U test, asterisks, for Kruskal–Wallis or Tukey’s HSD test, plus sign, for one-way ANOVA, p<0.05). Triangle indicates that seedlings were dead before 15 days after germination, so they were not measured. The cotyledon and first leaf length of S. densiflora were reduced as salinity increased in seeds sourced from every study location and elevational habitat (Figure 3). The length of cotyledon and first leaf were shorter for seedlings from LM seed that germinated at salinity higher than 0.30 M NaCl (Figure 3a). In contrast, seedlings from MM and HM seeds displayed this reduction in size at a lower salinity concentration of 0.15 M NaCl (Figure 3b,c). Also, in contrast, salinity had a positive effect on radicle growth at 0.30 M NaCl for seeds coming from LM, though radicle length was reduced at salinities higher than 0.45 M NaCl (Kruskal–Wallis test, H 5,205 =84.93, p<0.0001) (Figure 3a). This shorter radicle length trait was expressed in salinities higher than 0.30 M NaCl for seeds from MM (Kruskal–Wallis test, H 5,147 =28.99, p<0.0001) (Figure 3b) and higher than 0.15 M NaCl for seeds from HM (Kruskal–Wallis test, H 5,140 =26.65, p<0.001) (Figure 3c). In freshwater conditions, S. densiflora seedlings emerging from seeds sourced at Almendral and Bacuta locations had 1.3 times, and 1.7 times larger first leaves and radicles respectively, than those from Calatilla (Kruskal–Wallis test, H 2,111 = 10.17, p<0.01; one-way ANOVA test, F =4.88, df =2, p<0.01, respectively) (Figure 3d–f). Few significant differences, without showing a clear pattern, were recorded for seedling responses during the recovery experiment for all previous salinity exposures (Figure 3). Plants 2019,8, 372 7 of 15 Plants 2019, 8, 372 8 of 16 Figure 3. Cotyledon (white bars), first leaf (grey bars) and radicle length (black bars) from seedlings of Spartina densiflora germinated in six salinity treatments (left) and in the recovery assays after salinity exposure (R) (right) coming from seeds collected from three habitats (grouping the three locations in each habitat): (a) low marsh, (b) middle marsh, (c) high marsh, and from three locations (grouping the three habitats in each location): (d) Almendral, (e) Bacuta. (f) Calatilla), in the Odiel Marshes (Southwest Iberian Peninsula). Data show mean ± SE (n = 3–40). Different letters indicate significant Figure 3. Cotyledon (white bars), first leaf (grey bars) and radicle length (black bars) from seedlings of Spartina densiflora germinated in six salinity treatments (left) and in the recovery assays after salinity exposure (R) (right) coming from seeds collected from three habitats (grouping the three locations in each habitat): ( a ) low marsh, ( b ) middle marsh, ( c ) high marsh, and from three locations (grouping the three habitats in each location): ( d ) Almendral, ( e ) Bacuta. ( f ) Calatilla), in the Odiel Marshes (Southwest Iberian Peninsula). Data show mean ± SE (n =3–40). Different letters indicate significant differences among salinity treatments (Mann–Whitney U test, in italic, for Kruskal–Wallis or Tukey’s Plants 2019,8, 372 8 of 15 HSD test, in non-italic, for one-way ANOVA, p<0.05). Asterisks and plus sign indicate significant differences compared to control treatment (0.00 M) for each seedling parameter (Mann–Whitney U test, asterisks, for Kruskal–Wallis or Tukey’s HSD test, plus sign, for one-way ANOVA, p<0.05). 3. Discussion Our hypotheses were partially confirmed by the outcomes of the experiments. As expected, germination rates of seeds from native S. maritima expressed high tolerance to salinity, but tolerance of this native species was lower than that of salinity levels tolerated by invasive S. densiflora. In this circumstance, S. densiflora has a higher capacity for phenotypic plasticity in response to salinity. However, invasive seedling trait responses to salinity suggest a lower tolerance to salinity during the initial 15 days of seedling growth than the more vigorous initial growth of native S. maritima. Some salinity responses were common to both studied cordgrasses. For example, high seed viability after salinity exposure was recorded for both Spartina species, as it has commonly been observed for many halophyte species [ 9 ]. Also, elevated salinity concentrations inhibited and delayed germination for both studied Spartina species [ 8 ]. This seed quiescence prevents seed germination under stressful conditions [ 1 , 5 ]. Decreases in germination rates have been reported for the congener Spartina alterniflora Loisel. at salinities higher than 0.20–0.40 M NaCl in the native and invasive range [ 34 , 35 ], however some authors recorded high germination (>90%) even at hypersalinity [ 15 ]. Germination of Spartina ciliata Brongn. from Brazil was reduced at salinities higher than 0.20 M NaCl, totally inhibited at seawater concentration, and speed of germination increased after salinity exposure [ 36 ]. In addition, exposure to salinity followed by recovery after freshening accelerated germination for S. maritima and S. densiflora in our study, which has been reported previously for other halophyte species from a range of functional groups [ 8 , 37 – 39 ]. The observed stimulation of germination speed and rate after salinity exposure can provide windows of opportunity for seeds to germinate and quickly establish when salinity is sporadically and temporarily reduced by precipitation events or other sources of freshwater inflow [10,40]. While both studied cordgrasses showed high tolerance to salinity and shared some common germination and initial seedling trait responses, each also expressed distinctly contrasting responses. Germination rates and speed of germination under increasing NaCl concentrations indicated S. densiflora had higher germination tolerance to salinity than S. maritima.S. maritima germination was completely inhibited and S. densiflora was able to germinate at hypersalinity. Moreover, germination percentage decreased from 0.15 M NaCl up for S. maritima and from 0.30 M NaCl up for S. densiflora. As in our study, invasive S. densiflora in Humboldt Bay (California) showed reductions in seed germination at salinities higher than 0.30 M NaCl [ 30 ]. Some authors recorded total germination inhibition at 1.00 M NaCl and at 0.70 M NaCl for invasive S. densiflora in the Gulf of C á diz [ 29 , 31 ]. The great salinity tolerance of S. densiflora was also reflected on its rapid germination after being pre-treated at increasing salinities, whereas this study is the first to document the opposite response for native S. maritima. Seed quiescence of S. densiflora did not alter its initial seedling growth, in contrast to responses of S. maritima, in which seedlings were longer after saline pre-treatments compared to control. In addition to the capacity for seed quiescence under stressful conditions, the response of S. densiflora indicates a degree of physiological seed dormancy ( <10%). In addition, the radicle and first leaf of S. densiflora always emerged earlier than those of S. maritima. This suggests S. densiflora seeds were able to remain dormant in stressful saline environments without damaging the quality of the embryo, and then had the capacity to germinate later when salinity stress was reduced, providing multiple opportunities for establishment [1,10,39]. Its higher germination tolerance to salinity, faster germination after being pre-treated at increasing salinities and the presence of seed dormancy help to explain that S. densiflora is able to invade a wide range of habitats along the intertidal gradient [21] including hypersaline saltpans [3]. Plants 2019,8, 372 9 of 15 Invasive S. densiflora germination showed higher salinity tolerance than native S. maritima, but the autochthonous species showed higher salinity tolerance in relation to early seedling growth. Negative effects of salinity on seedling growth have been reported previously for invasive S. densiflora in Humboldt Bay (California) where seedling height decreased at salinities higher than 0.20 M NaCl [ 30 ], for invasive S. alterniflora in China [ 35 ], where shoot height decreased from 0.20 M NaCl up and radicle length from 0.10 M NaCl up, and for native S. ciliata in Brazil [ 36 ], with shoot and radicle being smaller at salinities higher than from 0.05 M NaCl. Besides the general comparison between both Spartina species, contrasted responses to salinity in seed viability, germination rate and speed, seed dormancy and seedling growth were also recorded among native and invasive species and among S. densiflora populations along the intertidal gradient. These differences in germination and seedling trait responses between S. maritima and S. densiflora and for seeds sourced among contrasting S. densiflora habitats could be attributed to local adaptation to contrasted environments [ 41 ], or to pre-adaptive conditioning determined by the maternal environment during seed development [ 42 – 44 ]. Salinity responses from different S. densiflora locations along the Odiel-Tinto Estuary (grouping LM, MM and HM elevations at each location) may support the pre-adaptive conditioning hypothesis. Supporting this idea, seed viability was high and independent of exposure to salinity concentrations for seeds from Almendral, but it decreased as salinity increased for seeds from Bacuta and Calatilla locations. Almendral is the nearest location to the coastline (12,500 m), whereas Bacuta and Calatilla are located along a tidal gradient 1800 m and 5500 m inland from Almendral, respectively. Thus, seeds ripening in low elevations and in locations closer to the coastline are more frequently exposed to tidal flooding and salt spray than those at higher elevations and more inland locations on the intertidal gradient [ 45 ]. Differentiated environmental conditions may acclimate seeds to salt stress in LM and closer to the sea, protecting their embryo from being killed due to ion toxicity at high salinities [ 2 ]. Furthermore, the invasive S. densiflora populations have low genetic diversity in North American and European marshes [ 46 , 47 ], which also supports that differences recorded along the intertidal gradient would likely be due to phenotypic plasticity rather than to genetic adaptation. Pre-adaptive conditioning determined by maternal stress conditions could increase survivorship and germination under high salinities, which may suppose an advantage for offspring in conditions similar to those experienced by the parents [ 42 – 44 ]. Moreover, salinity acclimation would facilitate survivorship during hydrochorus dispersal of buoyant seeds with sea water currents [ 48 ]. Other authors have observed that salt tolerance in halophyte germination is related to the duration and intensity of their exposure to salts in field conditions [ 9 , 11 , 32 , 49 ]. As in our study, Iris hexagona Walter and Suaeda aralocaspica (Bunge) Freitag and Schütze growing in high salinities produced seeds that had higher germination rates and speeds of germination when seeds were exposed to different salinity concentrations than seeds produced in low salinity environments [ 37 , 50 ]. Furthermore, other environmental factors such as temperature, photoperiod, soil moisture and nutrients availability can influence seed viability, germinability and dormancy [1,51–54]. 4. Materials and Methods 4.1. Study Area and Plant Material The plant propagules evaluated in this study were sourced from the Odiel Marshes by the Gulf of C á diz, in the Southwest Iberian Peninsula. The coast of the Gulf of C á diz is mesotidal and the mean sea level in this area is +1.85 m relative to Spanish Hydrographic Zero (SHZ). The tides are semidiurnal and have a mean range of 2.10 m and a mean spring tidal range of 2.97 m, representing 0.40–3.37 m above SHZ. This area is under a Mediterranean climate with Atlantic influence, with +18.2 ◦ C as the annual mean temperature [ 23 ]. Native vegetation in salt marshes along the Gulf of C á diz has been described in previous works [23,33,55]. Inflorescences in fruiting stage were randomly collected from S. maritima and S. densiflora tussocks in the Odiel Marshes. Since native S. maritima colonizes mainly low elevations in the tidal frame [ 23 ],