No evidence for local adaptation to salt stress in the existing populations of invasive Solidago canadensis in China

Local adaptation is an important mechanism underlying the adaptation of plants to environmental heterogeneity, and the toxicity of salt results in strong selection pressure on salt tolerance in plants and different ecotypes. Solidago canadensis, which is invasive in China, has spread widely and has recently colonized alkali sandy loams with a significant salt content. A common greenhouse experiment was conducted to test the role of local adaptation in the successful invasion of S. canadensis into salty habitats. Salt treatment significantly decreased the growth of S. canadensis, including rates of increase in the number of leaves and plant height; the root, shoot, and total biomass. Furthermore, salt stress significantly reduced the net photosynthetic rate, stomatal conductance, transpiration rate and relative chlorophyll content but significantly increased peroxidase activity and the proline content of S. canadensis and the root/shoot ratio. Two-way analysis of variance showed that salt treatment had a significant effect on the physiological traits of S. canadensis, except for the intercellular CO2 concentration, whereas the population and the salt × population interaction had no significant effect on any physiological traits. Most of the variation in plasticity existed within and not among populations, excep for the root/shoot ratio. S. canadensis populations from soil with moderate/high salt levels grew similarly to S. canadensis populations from soils with low salt levels. No significant correlation between salt tolerance indices and soil salinity levels was observed. The plasticity of the proline content, intercellular CO2 concentration and chlorophyll content had significant correlations with the salt tolerance index. These findings indicate a lack of evidence for local adaption in the existing populations of invasive S. canadensis in China; instead, plasticity might be more important than local adaptation in influencing the physiological traits and salt tolerance ability across the S. canadensis distribution.

Introduction tended to grow faster at a site of the highest elevation, whereas samples collected from warmer sites did not, indicating the possibility of local adaptation to cold weather [35]. Based on growth chamber and greenhouse experiments, we determined that the phenotypic plasticity of S. canadensis may have evolved rapidly in regions with different climatic conditions and might have contributed to the spread of this invasive species [22]. However, we also found that individual plasiticty, not local adaptation, plays an important role in the response of S. canadensis to shade [36]. Recently, S. canadensis was shown to be tolerant to salinity and to have invaded alkali sandy loams with a significant salt content, such as the Jiuduansha intertidal wetland shoals located at the junction of the Yangtze River and the East China Sea [37] and the polders on the wetland of Hangzhou Bay [38]. With regard to the toxic and selection pressure of salt, we hypothesized that local adaptation might play important role in the invasion success of S. canadensis into sodic soil. However, there is thus far no experimental evidence. Therefore, in this study, we conducted a greenhouse experiment with replicate cuttings of genets from different populations and addressed three questions: (1) Is the salt tolerance of S. canadensis populations related to soil salt levels? (2) Do S. canadensis populations from high salt-level soil grow better than those from low salt-level soil? If not, (3) does the variation in plasticity in response to salt exist within or among populations of S. canadensis? (4) Is the plasticity of physiological traits correlated with the salt tolerance of S. canadensis?

Plant species
Solidago canadensis, a forb belonging to the Asteraceae family, can produce annual clonal aboveground shoots from persistent belowground rhizomes [34]. This clonal growth leads to dense stands of shoots that reduce native species diversity [34]. The seeds are small, numerous, and wind dispersed, which is necessary for long-distance dispersal [34]. In addition, S. canadensis is self-incompatible [39].

Sample collection and propagation of plant materials
In the winter of 2012, rhizome systems were collected from 11 populations of S. canadensis in China. The populations came from representative habitats; most of the field sites were located near roads and consisted largely of ruderal vegetation. The field sites did not involve any endangered or protected species, and none of the populations were privately owned or under nature protection. No specific permissions were required for these locations. The geographical information and the types of land uses of the populations are shown in Table 1. Soil salinity levels were extracted from the Harmonized World Soil Database at a 0.5-arc-minute spatial resolution from FAO (http://www.fao.org/; detailed information is shown in Table 1). Excess free salts, referred to as soil salinity, is measured as electrical conductivity (EC) or exchangeable sodium percentage (ESP). Among the populations, the salinity levels of 7 were low (EC <4 ds/m or ESP < 6%), whereas those of the other 4 were moderate (EC = 4-8 ds/m or ESP = 6-15%), high (EC = 8-16 ds/m or ESP = 15-25%) or very high (EC > 16 ds/m or ESP > 15%) [40]. Within each population, 3 randomly selected shoot bases with attached rhizomes were dug up and kept moist until replanting. The clonal sprouts of S. canadensis generate from belowground rhizome fragments; thus, the distances between the shoots were at least 10 m to avoid sampling the same genet twice. All rhizome systems were planted in pots in a common garden at Taizhou University (E 121˚17´, N 28˚87´) in Linhai City, Zhejiang Province, China. The pots were 30 cm in diameter and 30 cm deep and were filled with a mixed matrix composed of soil, sand and peat soil in a 6:3:1 ratio with a final pH of 6.80±0.10, an organic matter content of 27.66±1.19 g/kg, a total nitrogen content of 361.00±33.00 mg/kg, an available phosphorus content of 8.00±1.14 mg/kg, and an available potassium content of 12.00±1.00 mg/kg. Flow cytometry analysis using a tender leaf showed that individuals from all localities sampled in this study exhibited the same ploidy level (i.e., hexaploidy; 6n = 54).

Salt treatment experiment
The salt treatment experiment was conducted in a greenhouse at Taizhou University under identical light, humidity and temperature conditions in the summer of 2014. The mean daily temperature ranged from 25˚C to 34˚C. The photosynthetic active radiation is approximately 80% of the strength of natural sunlight. Rosettes with a similar height (mean = 15 cm) were selected and removed from the collected rhizome systems after sprouting and individually planted in pots (16 cm diameter, 14 cm height) containing the mixed matrix. For every population, three genotypes were used as replicates. For every genotype, two rosettes were used for the salt treatment and control. One rosette was planted in each pot. All plant materials used in the different treatments in this study were clonally propagated two years after being commonly planted so that the differences caused by different genotypes and the effect of the mother body were excluded in the experiment. Eight days after planting, 50 ml of 1/8 Hoagland solution was applied to each pot. Ten days after planting, the plant height (H t1 ) and number of leaves (N t1 ) were calculated as the initial data (t1 = 0 day). Plant height was recorded as the distance from the ground to the highest leaf position. To set up the periodic salt treatments, 50 ml of a 300 mmol/L NaCl solution was applied to each pot for two days, followed by 50 ml of tap water for one day, then 50 ml of Hoagland solution for one day. For the control, 50 ml of tap water was applied to each pot for three days, followed by 50 ml of Hoagland solution for one day. The positions of the pots were randomized within the experimental chamber every week to confirm that no position effect occurred.

Measurements
Twenty-eight days after salt treament, plant height (H t2 ) was measured via ruler with an accuracy of 0.1 cm. The number of leaves (N t2 ) was also recorded. The increase rate in plant height was calculated as (H t2 -H t1 )/(t 2 -t 1 ) and the rate of increase of the number of leaves was calculated as (N t2 -N t1 )/(t 2 -t 1 ).
In situ photosynthesis measurements were made on the third fully expanded leaf, using a portable photosynthesis-measurement system (LI-6400 XT, Li-COR Inc., Lincoln, NE, USA). Measurements were obtained between 9:00 AM and 11:00 AM under a photosynthetically active radiation of 1,400 μmol m -2 s -1 (i.e. at light saturation) at a leaf temperature of 25˚C, a CO 2 concentration of 400 ppm, and relative humidity of 70%. Net photosynthetic rate (P n ), stomatal conductance (g s ), transpiration rate (T r ), and intercellular CO 2 concentration (C i ) were measured.
Three tender leaves per plant were collected and transferred to the lab immediately upon collection. The content of proline was determined via acid ninhydrin colorimetry using L-proline as the standard [41]. The activity of peroxidase was measured by ultraviolet spectrometry [42]. The relative chlorophyll content was measured using a chlorophyll content meter (CCM-200 plus, Opti-Science Inc. Hudson, NH, USA).
Following measurements, plants were harvested and divided into leaves, stems, and roots. Plant material was over-dried (at 105˚C for 1 h and then at 80˚C until a constant weight was reached). The leaf, stem, and root biomasses were weighed using a balance with a precision of 0.0001 g (Shanghai Jingtian Electronic Instrument Co., Ltd, Shanghai, China). Total biomass and root/shoot ratios were calculated.

Statistical analyses
The tolerance index (TI) in terms of the root, shoot, total biomass and plant height was calculated for each population, i.e., the index of the plants under salinity stress divided by the index of the plants in the control [43]. The phenotypic plasticity index was calculated based on the difference between the maximum mean and minimum mean divided by the maximum mean [44].
All data are shown as the mean ± standard error. A t-test was applied to test the effect of the salt treatment on the plants. A two-way analysis of variance (ANOVA) was applied to test the effects of salt, population and their interaction on physiological traits, with the treatment as a fixed factor and the population and genotype (nested to population) as random factors. Oneway ANOVA was applied to test the differences in plasticity among different populations. A variance component analysis was conducted to calculate the differentiation coefficient of the phenotypic plasticity index within or between different populations. The relationships between phenotypic plasticity and the salt tolerance index were evaluated separately by Pearson's correlation analysis. The relationships between the salt tolerance index and soil salinity level were evaluated using Spearman's rho value through a nonparametric test (binomial test). All statistical analyses were conducted using SPSS 16.0 software.

Effects of salinity on growth and salt tolerance
The salt treatment significantly inhibited the rate of increase in the number of leaves (Fig 1A, paired t value = 8.987, p<0.001) and the rate of increase in plant height (Fig 1B, paired t  value = 7.245, p<0.001). Salt treatment also significantly reduced the root biomass (Fig 2A, paired t value = 4.306 p = 0.002), shoot biomass (Fig 2B, paired t value = 6.597, p<0.001) and total biomass (Fig 2C, paired t value = 6.733, p<0.001). Under salt treatment, the root, shoot, and total biomass and plant height of S. canadensis populations from moderate/severe saltlevel soil were similar to those of S. canadensis populations from low salt-level soil (Fig 3).
Based on the root, shoot and total biomass, the salt tolerance of S. canadensis of the NT population was highest, whereas the salt tolerance of the LYG population was lowest. Based on plant height, the salt tolerance of the HZ and TZ populations was highest and lowest, respectively (Table 2). However, no significant differences were found among the populations (p>0.05). Nonparametric correlations analysis showed no significant correlation between salt tolerance indices and soil salinity levels ( Table 2).  Effects of salinity on physiological traits, root/shoot ratio and their plasticities The salt treatment significantly increased peroxidase activity, proline content and root/shoot ratio, and significantly decreased the net photosynthetic rate, stomatal conductance, and transpiration rate; however, the salt treatment had no significant effect on the intercellular CO 2 concentration (Table 3). Two-way ANOVA showed that the salt treatment had a significant effect on the root/shoot ratio and the physiological traits of S. canadensis, except for the intercellular CO 2 concentration; In contrast, the population, genotype and the interaction salt × population had no significant effect on all traits. The proline content showed the strongest plasticity, whereas the intercellular CO 2 concentration and root/shoot ratio showed the weakest plasticity (Table 3). Most of the variation in plasticity existed within populations and not among populations, except for the root/shoot ratio (Table 3).

Correlation between the salt tolerance index and the plasticity of physiological traits and the root/shoot ratio
Pearson's correlation analysis revealed significant correlations between the plasticity of proline content and the salt tolerance index based on plant height; the plasticity of the intercellular CO 2 concentration and salt tolerance indices based on total biomass, shoot biomass and plant height; and the plasticity of the chlorophyll content and the salt tolerance index based on plant height (Table 4).

Discussion
Salinity is a limiting environmental factor that impairs plant growth and development [45]. The toxicity of salt results in strong selection pressure on the salt tolerance of plants and different ecotypes, and genetic mutants with salt tolerance have evolved during the adaption to salt-affected soil [46]. In our greenhouse experiment, we did not find that S. canadensis from higher saline-level soil performed better than plants from low saline-level soil; furthermore, the salt tolerance indices in terms of root, shoot, total biomass and height did not show any significant correlations with the soil salinity levels. Our results suggest the absence of local adaption in the existing populations of S. canadensis collected from soils with medium, high and extremely high salinity, which was contrary to our original hypothesis. Similar phenomena have been observed in the perennial clonal plant Leymus chinensis in response to water [7] and the invasive plant Fallopia japonica in response to salt [3], but not in invasive Ipomoea cairica in response to salt [47] and invasive S. canadensis in response to cold [35]. Richards et al. hypothesized that local adaptation would occur in F. japonica established in salt marsh habitats because of increased salt tolerance compared to those established in the more common roadside habitat. However, these authors found that F. japonica from the salt marsh habitats did not perform better in the salt treatment, suggesting that selection by the salt content of the marsh habitat did not generate genotypes adapted to high salinity [3]. Having conducted a meta-analysis of local adaptation in plants, Leimu and Fischer suggested that local adaption is less common in plant populations than is generally assumed [48]. The occurrence and strength of local adaptation by plants in response to environmental variation may be dependent on species as well as on the life history, population size, and study characteristics [49]. The absence of local adaptation of S. canadensis in sodic soils might be due to three reasons. The first is the extremely high plasticity of S. canadensis, which might cover the effect of genotype variation. The second is the relatively short invasion history in colonization of sodic soil. Although differences in soil salinity levels existed in the different population, the short invasion history and weak selection pressure might not have contributed to the local adaptation of S. canadensis populations. The third is the population origin and non-quantitative measurment of soil salinity, which might also be the main limitation of the experiment design. The populations used in the study were collected from normal habitats and, not from coastal areas, where the difference in soil salinity levels might be greater. In addition, we extracted the soil salinity level data from the Harmonized World Soil Database at a 0.5-arc-minute spatial resolution from FAO, which might not be consistent with the local habitat conditions from which the S. canadensis individuals were collected. The relatively small difference in soil salinity levels and the relatively coarse broad-scale information of soil salinity levels might have affected the corrlation between salt tolerance indices and soil salinity levels. The existence of local adaption to cold temperature has been reported [35]. Thus, although no clear evidence for local adaptation was found in this study, we should still be careful in drawing a general conclusion about  Salt response of Solidago canadensis the absence of local adaption of S. canadensis in colonization of sodic soils. Further study should involve collection of plants from sodic soils with clear and relatively greater differences in salinity levels, direct measurement of the soil salinity content and calculation of the relationship between salt tolerance indices and the quantitative sat content. Plasticity for ecologically important traits may enhance the ability to withstand adverse environmental conditions or to respond positively to favorable conditions, thus promoting invasiveness [49]. It has been well documented that phenotypic plasticity may be a common trait of plant invaders (reviewed by Richards et al. [19]. Richards et al. demonstrated that plasticity in salt tolerance traits might allow invasive Fallopia japonica and F. × bohemica to live in saline habitats without any specific adaptation to tolerate salt [3]. Our previous study showed that the phenotypic plasticity of S. canadensis is high in response to temperature and water availability [22]. In the present study, we observed strong plasticity of the proline content, intermediate plasticity of the net photosynthetic rate, stomatal conductance and relative chlorophyll content, and relatively weaker plasticity of the transpiration rate and the activity of peroxidase. Pearson's correlation analysis revealed a significantly negative correlation between the plasticity of proline content or chlorophyll content and the salt tolerance index based on plant height and a significant positive correlation between the plasticity of the intercellular CO 2 concentration and salt tolerance indices based on total biomass, shoot biomass and plant height. Our experimental evidence suggests that phenotypic plasticity may be the primary adaptive strategy for invasive S. canadensis with regard to salinity tolerance. These results show that the ability of S. canadensis to maintain higher chlorophyll content, lower proline content and higher intercellular CO 2 concentration enables greater growth at higher levels of salinity.
Although the variation of phenotypic plasticity among invading populations has rarely been documented [50], some studies have indicated that variation of plasticity among introduced populations may allow an invader to evolve greater plasticity, resulting in the colonization of more diverse habitats and plant communities [17,49,50]. A previous study showed that individual plasticity, not local adaptation, played a more prominent role in the shade response of invasive S. canadensis populations under similar light conditions [36]. In the present study, we found that most of the variation in the plasticity of the physiological traits of S. canadensis occurred among individuals within populations and not among populations, indicating that the potential of S. canadensis to evolve greater tolerance or adaptation to saline soil was small. Similar results were obtained for the clonal perennial plant Leymus chinensis [7] but not the invasive plant Microstegium vimineum [49] and Ipomoea cairica [47]. In addition, we also found taht most of the variation in the plasticity of the root/shoot ratio of S. canadensis occurred among populations, indicating that the evolutionary potential of the variation in root/shoot ratio was large. However, we did not find a significant correlation between salt tolerance indices and the plasticity of the root/shoot ratio, which might also be due to the short colonization history of S. canadensis in saline habitats or the small difference in soil salinity levels.
Taken together, these findings indicate that individual plasticity might be more important than local adaptation in affecting physiological traits and salt tolerance across the distribution of the species. We predict that S. canadensis, which is dominant on broadsides [34], might have the potential to become a serious problem in sodic soils, including coastal areas, in the future. More attention should focus on monitoring the occurrence of S. canadensis on sodic soils, and timely prevention should be implemented to control new invaders.

Author Contributions
Conceptualization: JL MY.