Introduction

Invasive species of aquatic macrophytes often promote the homogenization of habitats, reduction the diversity species and life forms (submerged, free floating, emergent) of aquatic macrophytes (Amorim et al., 2015; Thomaz et al., 2015). These invaders may also negatively affect other aquatic communities, reducing the richness of fish species (Carniatto et al., 2013), modifying the composition of the associated fauna (Houston & Duivenvoorden, 2002; Midgley et al., 2006; Mormul et al., 2010; Coetzee, 2014) and decreasing the abundance of phytoplankton (Villamagna & Murphy, 2010).

In estuaries, the introduction of exotic species is of major concern, since these ecosystems have high biodiversity and are important nursery grounds for several species (Odum, 1988; Levin et al., 2001). The great diversity and richness of species in these ecosystems derive from the longitudinal gradients of salinity, flooding and nutrient concentration due to the influence of seawater (Ribeiro et al., 2011; Nunes et al., 2019). These longitudinal gradients are decisive in the distribution of aquatic macrophyte species. In tropical estuaries, there is a marked longitudinal salinity gradient, with low salinity in the upper estuary (predominance of fresh water), intermediate salinity in the middle estuary, and high salinity in the lower estuary (predominance of salt water) (Nunes & Camargo, 2018). Thus, salinity is an important variable to determine the distribution of aquatic macrophytes and the colonization by invasive macrophytes (Nunes & Camargo, 2018). For instance, conditions of high salinity may prevent the expansion of freshwater macrophytes (Thouvenot & Thiébaut, 2018), while selecting individuals and increasing the possibility of invasion by species adapted to saline environments (Xue et al., 2018). Furthermore, the increase in salinity in response to sea level rise due to climatic changes can affect coastal plant communities to a greater or lesser extent, depending on location, conditions and plant tolerances (Short et al., 2016), which increases the importance of studies on estuarine regions. Salinity is the main abiotic factor that limits the development and primary production of plants (Houle et al., 2001; Esteves & Suzuki, 2009), since it may cause ionic disequilibrium and oxidative stress (Esteves & Suzuki, 2009). However, some species have the ability to adapt and/or tolerate salinity, as a result of different types of adjustments, such as osmotic (Flowers & Colmer, 2008), morphological (Esteves & Suzuki, 2009), physiological (Larcher, 2000) and biochemical (Gratão et al., 2005).

Oxidative stress is an important indicator of salt stress, since the plant under the effect of salt may increase the production of reactive oxygen species (ROS) and suffer cell damage (Koyro et al., 2013; Gil et al., 2020). Among the ROS, hydrogen peroxide (H2O2) is one of the main cellular metabolites that, when at low concentrations, acts as an important signaling factor in the cell defense metabolism (Gill & Tuteja, 2010; Hussain et al., 2015; Foyer et al., 2017). On the other hand, H2O2 at high concentrations and in the presence of transition metals may generate the hydroxyl radical (OH⋅) that is capable of transposing and disintegrating cell membranes (Barreiros et al., 2006), forming small fragments of hydrocarbons, such as ketones, malondialdehydes (MDA), among other products related to lipid peroxidation (Garg & Manchanda, 2009; Halliwel & Gutteridge, 2015). In order to deal with the effects of ROS, an enzymatic complex (Foyer & Noctor, 2005; Gratão et al., 2012) and a non-enzymatic antioxidant mechanism (Carvalho et al., 2010; Foyer & Noctor, 2013) act in cell detoxification, preventing severe cell damage (Gratão et al., 2005; Hafsi et al., 2010), allowing the plant to grow in places with greater salinity.

Urochloa arrecta (Hack. ex Durand & Schinz) Morrone & Zuloaga is an invasive plant species originally from Africa, which occurs in all regions of Brazil (Flora of Brazil, 2020) and has been observed in different aquatic ecosystems, such as in the wetlands of the State of Mato Grosso (Pott et al., 2011), reservoirs (Michelan et al., 2010b; Rodrigues et al., 2017), coastal rivers and lakes (Amorim et al., 2015; Ferreira et al., 2017). Although this species has a preference for freshwater environments, U. arrecta forms dense and extensive stands in estuaries in the south and southeast of Brazil (Reinert et al., 2007). U. arrecta, like other Poaceae species, has salt glands (Bora et al, 2020), which explains its occurrence in estuarine areas.

Despite its importance, only two studies have evaluated the distribution of U. arrecta in estuaries and obtained salinity data at their place of occurrence. Nunes et al. (2019) observed this species in the upper estuary, in places of freshwater, while Bora et al. (2020) observed stands of this species in a middle estuary with salinity of approximately 3 ppt. These latter authors observed that this species grows at intermediate levels of salinity and develops roots and shoots also at 4.0 ppt of salinity, in which mangrove vegetation occurs. This demonstrates that populations of U. arrecta exposed to salinity are more resistant to high salinity. In the southeastern and southern estuaries of Brazil, the native aquatic macrophytes Spartina alterniflora Loisel (Poaceae) and Crinum americanum L. (Amaryllidaceae) are the main species that occur along a longitudinal salinity gradient (lower to upper estuary). In general, S. alterniflora occurs in the lower estuary (salinity from 20 to 30 ppt), C. americanum occurs in the upper estuary (salinity from 0 to 5 ppt) and both species form mixed stands in the middle estuary (salinity from 5 to 20 ppt). Our question is: Can U. arrecta expand to the middle and lower estuary areas, occupying the space of the native species and affecting areas critical for conservation?

Understanding the distribution and potential expansion of an invasive species depends on the adaptation and response mechanisms to a stressful condition. Since the increase in oxidative stress and the reduction in growth are effects that the plant may present in the presence of salt stress, we made an experiment with the aim of evaluating the effect of the salinity in low estuary areas (20 and 30 ppt) on biomass gain and on the H2O2 and MDA contents of U. arrecta. Our hypotheses regarding the responses of U. arrecta at the three levels of salinity tested are that: (i) at the salinity of 0 ppt U. arrecta will present greater biomass gain and less production of H2O2 and MDA; (ii) at 20 ppt U. arrecta will present reduced biomass gain and H2O2 production, and intermediate MDA; (iii) at 30 ppt U. arrecta will not grow and will have a large production of H2O2 and MDA.

Methods

Species and sampling area

The species U. arrecta is an emergent aquatic macrophyte belonging to the Poaceae family. This species is originally from Africa and has been previously described as U. subquadripara (Thomaz et al., 2009; Michelan et al., 2010b; Alves et al., 2017). It is invasive and common in several areas of humid ecosystems (Pott et al., 2011; Amorim et al., 2015). U. arrecta has roots fixed on the margins and floating stems (nodes + internodes + leaves = stems) that extend to limnetic regions (Michelan et al., 2017). It is a perennial and stoloniferous aquatic macrophyte, with insignificant seminiferous propagation, and fast growing from fragments (Michelan et al., 2010a). On the Itanhaém River, which is located on the South coast of the State of São Paulo (Brazil) (23° 50′ and 24° 15′ S; 46° 35′ and 47° 00′ W), the stands of U. arrecta are distributed in the upper and middle area of the estuary (Umetsu et al., 2018).

Experiment

From September to December 2019 we conducted an experiment at the Faculty of Agricultural and Veterinary Sciences, UNESP—São Paulo State University, Jaboticabal, SP, Brazil (21° 14′ 05″ S, 48° 17′ 09″ W, and 615.01 m of altitude). The climate classification is Aw (tropical) according to Köppen and Geiger. The minimum and maximum temperatures in the period of the experiment were respectively 19.5°C and 32.6°C. The intensity of light (hours) and global solar radiation (Wh m−2) was respectively 220.6 and 20.6 (FCAV/UNESP Agroclimatological Station—Jaboticabal Campus, Brazil).

We performed a completely randomized single-factor experiment (1 species × 3 salinities × 5 repetitions = 15 experimental units) to assess U. arrecta growth and oxidative stress at different levels of salinity (0, 20 and 30 ppt). The experiment lasted 121 days and was conducted in a greenhouse (Fig. 1). The initial density was 8 macrophytes per experimental unit, based on Nunes and Camargo (2018, 2020). We considered each U. arrecta stem as an individual, that is, each clonal emergence above the substrate.

Fig. 1
figure 1

Representative scheme of the experimental units distribution in a greenhouse

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The U. arrecta individuals used in this experiment were composed of stem fragments with three nodes (approximately 15 cm) that were taken from a population of a monospecific stand of U. arrecta in the upper estuary of the Itanhaém River. The individuals were washed to remove materials adhered to the fragments and planted (approximately 3 cm deep inside the substrate) in the experimental units. As the substrate for the development of the aquatic macrophyte, we used a volume of 13 l of fine-grained washed sand (grains from 0.05 to 0.42 mm) in plastic boxes with a volume of 26 l and an area of 0.13 m2. After planting, the aquatic macrophytes were kept in acclimatization for 31 days with addition of 700 ml of modified Hoagland nutrient solution (Hoagland & Arnon, 1950; Mendelssohn et al., 2001) in each experimental unit every 15 days. From the 31st day, 2 l of saline solution and 700 ml of the same nutrient solution were added to the experimental units. The saline solutions consisted in 20 g l−1 and 30 g l−1, respectively, in the treatments with salinity of 20 and 30 ppt. The salt concentrations we used were based on the salinity of the sediments of the middle and lower estuary of the Itanhaém River, places where the native species S. alterniflora and C. americanum occur. For the saline solutions we used formulated sea salt (Aquaforest Sea Salt) which, in addition to sodium chloride, contains sulfates, calcium, potassium, magnesium and other minerals found in seawater (Nunes & Camargo, 2020). In the treatment of 0 ppt, in substitution to the saline solution, 2 l of tap water were added. In addition, we added tap water whenever necessary to maintain the water level of the boxes. We controlled the salinity level in the sediment every 15 days by placing 15 g of fresh substrate in 500 ml of distilled water, and measuring the salinity (ppt) of the solution using the Horiba equipment, model U-50. The salinity mean values and standard deviation (SD) in each treatment throughout the experiment were: 0 ppt (without variation), 19.6 ppt (SD = 2.81) and 29.8 ppt (SD = 3.78). The salinity remained significantly different (P < 0.05) among all treatments throughout the experiment.

Biomass and nutrient concentration

At the beginning of the experiment and at intervals of 15 days, we performed measurements of the length (meter) of the emergent fraction of each individual to estimate the aboveground biomass (AB) during the experiment and obtain the growth curves of the emergent fraction of U. arrecta. To obtain AB values, we used the non-destructive method proposed by Nunes and Camargo (2017). We estimated the aboveground fraction dry weight by a simple linear regression equation between height and dry mass (Eq. 1). The models we found were

$${\text{AB}} = \left( {2.286*h} \right) - 0.4773\, \left( {R^{2} = 0.7974} \right)$$
(1)

where AB is the aboveground biomass and \(h\) is the height of each individual in meters. The numbers in the equation correspond to the slope of the line and the Y-intercept. We calculated the gain of aboveground biomass—GAB (biomass of day 121 minus biomass of day 1) in each experimental unit and applied an analysis of variance (ANOVA) to verify the occurrence of significant differences among the treatments. We considered the initial biomass of the belowground fraction equal to zero. Thus, the gain of belowground biomass (GBB) was calculated from the dry mass obtained at the end of the experiment. At the end of the experiment, we separated the aboveground fraction (stems) and the belowground fraction (roots) of U. arrecta individuals from each experimental unit and washed them to remove any adhered material. After washing, we collected approximately 10 g of leaves (green and healthy) and roots (healthy) that were then wrapped in aluminum foil and immediately inserted in liquid nitrogen at the collection site. Subsequently, these samples were stored in a freezer at − 80°C for subsequent analysis of the content of MDA and H2O2 in the laboratory. The remaining aboveground and belowground fractions were dried in an oven at 60°C and ground in a mill. From the powdered plant material, we determined the total nitrogen (TN) content by the Kjeldahl method and total phosphorus (TP) (Allen et al., 1974) in aboveground and belowground fractions in the laboratory.

Oxidative stress indicators

The content of hydrogen peroxide (H2O2) was measured according to the method of Alexieva et al. (2001). We used the leaves (0.8 g) and roots (1 g) stored at − 80°C for the extraction. The absorbance was read at 560 nm and the results were expressed in µmol g−1 of fresh weight.

Lipid peroxidation was determined by the production of metabolites that are reactive to 2-thiobarbituric acid (TBARS), mainly MDA, according to the method of Heath and Packer (1968). The extraction followed the same protocol for H2O2. The concentration of MDA equivalents was measured by spectrophotometry between 535 and 600 nm; the results were obtained using an extinction coefficient of 1.55 × 10−5 mol−1 cm−1 (Gratão et al., 2012).

Statistical analysis

The salinity levels (0, 20 and 30 ppt) were used as predictor variables and the variables of GAB, GBB, TN, TP levels and stress indicators (H2O2 and MDA) as response variables. First, we submitted the data (GAB, GBB, TN, TP, H2O2 and MDA) to the Cramer–Von–Misses and Bartllet test, to verify the normality of the residual and the homoscedasticity of data variance, respectively. After meeting these premises, the analysis of variance (ANOVA) was used to verify the occurrence of significant differences among the treatments. When this difference was statistically significant (P < 0.05), the Tukey’s test was applied for the multiple comparison of the means with a 95% confidence interval. Statistical analyses were performed using the software R, version 3.4 (2017), and the graph and figures were prepared using Microsoft Excel® (2010).

Results

The GAB of U. arrecta was significantly higher at 0 ppt, intermediate at 20 ppt and lower at 30 ppt, while the GBB was significantly higher at 0 ppt and lower at 20 and 30 ppt (Fig. 2; Table S1). The growth curve of the aboveground fraction of U. arrecta was similar for the different treatments up to day 46, and on day 121 of the experiment, the growth was higher at 0 ppt, intermediate at 20 ppt and lower at 30 ppt (Fig. 3). The content of TN (%) in the aboveground fraction of U. arrecta showed no significant difference among the treatments. Nonetheless, in the belowground fraction, the TN content was greater at 20 and 30 ppt than at 0 ppt. The TP content (%) of the aboveground fraction was significantly higher at 0 ppt and lower at 30 ppt. However, in the belowground fraction the TP content was significantly higher at 20 and 30 ppt and lower at 0 ppt (Table 1).

Fig. 2
figure 2

Mean and standard deviation values of A gain of aboveground biomass (GAB), and B gain of belowground biomass (GBB) of U. arrecta in salinities of 0, 20 and 30 ppt. Bars followed by different letters are statistically different P ≤ 0.05 (Tukey’s test)

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Fig. 3
figure 3

Growth curves of the aboveground fraction of U. arrecta (bars = standard deviation values) in salinities of 0 (continuous line), 20 (dashed line) and 30 (dotted line) ppt

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Table 1 Mean values (standard deviation) and results of the ANOVA for TN and TP in the aboveground and belowground of U. arrecta in 0, 20 and 30 ppt salinities
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The H2O2 content (µmol g−1 of fresh mass) was higher in the aboveground fraction of U. arrecta in the treatments with salinity of 20 and 30 ppt than at 0 ppt. Nevertheless, the MDA content (nmol g−1 of fresh mass) was higher in the treatment with salinity of 30 ppt than at 0 and 20 ppt, indicating that salinity induces a greater production of reactive oxygen species (ROS) at both concentrations, leading to lipid peroxidation in aquatic macrophytes submitted to higher salinity. However, in the belowground fraction of U. arrecta, there was no significant difference in the contents of H2O2 and MDA among the salinity levels (Fig. 4; Table S2), indicating that salinity did not induce oxidative stress in the belowground fraction.

Fig. 4
figure 4

Mean and standard deviation values of A H2O2 (µmol g−1 of fresh weight) and B MDA (nmol g−1 fresh weight) contents in aboveground (light gray) and belowground (dark gray) of U. arrecta in salinities of 0, 20 and 30 ppt. Bars followed by different letters are statistically different P ≤ 0.05 (Tukey’s test)

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Discussion

The results of our experiment showed that salinity limits the growth and biomass gain of U. arrecta, which corroborates our initial hypothesis. This result is similar to those obtained by other authors. Bora et al. (2020) observed that salinity was stressful for U. arrecta, reducing the growth and biomass gain of individuals from both freshwater and brackish water populations. Other emerging species tolerant to salinity, such as Distichlis spicata (L.) Greene (Poaceae) and Scirpus robustus Pursh (Cyperaceae), also had a reduction in aerial biomass in response to salinity (Crain et al., 2004).

A possible explanation for this response could be related to the high loads of energy expended by the plant to adjust to salt stress (Esteves & Suzuki, 2009). In fact, mangrove species need to maintain continuous water absorption, in addition to regulating ionic absorption and compartmentalization against a strong external salt gradient (Ball, 1996; Krauss et al., 2008). To maintain water absorption during salt stress, plants do not only need to reduce water loss by morphological and physiological adaptations, but they also need to keep water potentials sufficiently low (Krauss et al., 2008). In Poaceae species, the excretion of salt through the salt glands plays an important role in the regulation of ionic balance, contributing to tolerance to salinity (Manousaki & Kalogerakis, 2011; Céccoli et al., 2015; Atia et al., 2019). Nevertheless, salt excretion is an energy-dependent process that moves ions against large electrochemical potentials within the leaves (Lüttge, 2007). The removal of excessive salt also occurs by the translocation of ions to older leaves, which consequently promotes leaf senescence (Cram et al., 2002; Munns & Tester, 2008).

Therefore, a shorter life expectancy may accelerate leaf renewal, increasing the proportion of young leaves with a high photosynthetic rate and reducing the cost associated with maintaining older leaves. In addition, under natural conditions, variations in sediment salinity over time contribute to reactivate growth after prolonged periods of hypersaline conditions, preventing plant death (Suárez & Medina, 2005). In this sense, it is possible to assume that the lower growth and GAB of U. arrecta in treatments with 20 and 30 ppt derives from factors such as higher energy demand for mechanisms of salt tolerance, high rate of leaf senescence and absence of variation in salinity throughout the experiment.

Plant species tolerant to salinity also need a well-adjusted mechanism to combat the effects of oxidative stress promoted by salinity (Esteves & Suzuki, 2009; Koyro et al., 2013; Gil et al., 2020). In our experiment, salinity promoted increased ROS production, observed by the higher H2O2 contents, which corroborates our hypothesis. Nonetheless, regarding lipid peroxidation in the treatments with salinity of 20 and 30 ppt, our hypothesis was corroborated for individuals at salinity of 30 ppt, but not for individuals at salinity of 20 ppt. This answer shows us that, at high salinity, stress was greater, and the antioxidant mechanism was less efficient.

At intermediate salinity, the antioxidant mechanism of the aquatic macrophyte was more efficient in combating the effects of H2O2 in the cell. There is a close relationship between ROS production and reduced plant growth during salt stress. Koyro et al. (2013) identified, in the species Panicum turgidum Forssk. (Poaceae) exposed to salinity, an increase in H2O2, reducing the activity of the Calvin cycle, as well as carbon fixation and the aerial biomass of the aquatic macrophyte. Salinity affects photosynthesis because, during saline stress, the plant’s ability to absorb water from the sediment is reduced and stomatal closure is triggered to reduce water losses in the form of evaporated water (Ahanger et al., 2017). Stomatal closure promotes a series of reactions that facilitate the formation of ROS, a reduction in the photosynthetic rate that compromises CO2 availability for carboxylation reactions (Munns & Tester, 2008; Esteves & Suzuki, 2009; Parihar et al., 2015) and plant growth (Li et al., 2019). Thus, contrarily to U. arrecta individuals under 30 ppt of salinity, individuals present at 20 ppt are probably exposed to lower selective pressure and less stress, which could be attributed to the greater efficiency of the mechanism responsible for the elimination of ROS, allowing the growth and lower biomass gain of this species under this condition.

In the belowground fraction, the absence of difference in the contents of MDA and H2O2 among the treatments indicates that the oxidative stress in this fraction was lower than in the aboveground fraction and, probably, this was not one of the factors responsible for the reduction in GBB. Although salinity promotes hypoxia in the soil, reducing transpiration, nutrient absorption and, consequently, plant growth (Morard & Silvestre, 1996), in the present study, the N and P contents of the individuals were not affected by salinity. A possible explanation for the reduction in biomass in the belowground fraction may be related to the strategy that some species adopt to combat salinity, and it may be due to root elongation. Root elongation at a higher salinity may assist in sediment aeration and minimize the negative effects of salt stress (Li et al., 2019), and consequently facilitate nutrient uptake by the roots. Although root length was not assessed in our study, Bora et al. (2020) observed that in treatments with higher salinity, U. arrecta showed greater root elongation at the expense of biomass allocation, probably as a salt tolerance strategy.

The adoption of strategies that also help the plants in nutrient absorption are important, because when a species is under saline stress, the physiological requirement for N is usually higher (Crain, 2007; Nunes & Camargo, 2020). Crain (2007) observed that Spartina patens Muhl. (Poaceae) present in swamps with high salinity levels showed a higher percentage of N in their tissues, which may be related to the increasing demand for N to help with salinity tolerance and not due to the availability of N in the sediment, since these environments tend to have less availability of this nutrient. Nunes & Camargo (2020) observed that C. americanum, when cultivated in sediments with a salinity of 20 ppt, has a higher N content in the emerging fraction. One explanation for this higher N content is the result of an osmotic adjustment to maintain a positive turgor pressure (Flowers et al., 2015). Nitrogen compounds are an important and efficient solute to adjust ion balance in vacuoles and promote the osmotic adjustment of the plant (Esteves & Suzuki, 2009). In this sense, it is possible that the higher absorption and concentration of N in U. arrecta, in the treatments with salinity of 20 and 30 ppt, is also a strategy of the aquatic macrophyte to adjust osmotically and tolerate the salt.

Although in our experiment U. arrecta tolerated the stress and grew in 20 ppt of salinity, in the Itanhaém river estuary, this does not occur in the area with this salinity in the sediment. Probably, other biotic factors, e.g. competition, are responsible for the absence of this invasive species in the middle estuary area. Some studies attribute the absence of freshwater species in salt swamp regions to salt stress (Crain et al., 2004; Engels & Jensen, 2010). However, Nunes & Camargo (2018) demonstrated that competition in saline environments may also be a limiting factor for a freshwater species. In addition, the lower areas of the estuaries are more subject to variations in the water level because of their proximity to the ocean, which can also be a stress factor for some emerging species (Deegan et al., 2007; Ribeiro et al., 2011).

Rising water levels in the oceans due to climate change will affect the distribution of aquatic macrophytes in estuaries. In fact, the sixth report by the IPCC Working Group I shows that there will be a temperature increase of at least 1.5°C over the next two decades. Between the years 1901 and 2018, the global mean sea level increased by 0.20 m and by 2100 it is estimated that these levels may exceed 1 m if carbon emissions are not reduced (IPCC, 2021). According to Callaway et al. (2007), the increase, however small, in salinity due to sea level rise can lead to changes in the distribution of estuarine species, with freshwater swamps being replaced by brackish swamps and brackish swamps converted to salt marsh communities. This will imply the reduction of freshwater areas (high estuary) and may increase the competition of U. arrecta with native species. The saline intrusion in the freshwater areas of the upper estuary may select salinity-tolerant populations of U. arrecta that will occupy areas currently colonized by native species tolerant to brackish waters, such as C. americanum and S. alterniflora.

Based on the results of our experiment, we conclude that U. arrecta develops physiological adjustments that allow its survival in a salinity of 20 ppt (medium estuarine sediment salinity), as the cell stress is controlled and the GAB was greater than 60% with the increase in salinity. It is important to note that only biomass gain and oxidative stress responses of U. arrecta in two salinities are not sufficient to predict the invasiveness of the species. Other abiotic and biotic factors are also important with regard to the successful invasion of a particular species. Thus, this study is only an indication that U. arrecta is probably able to adjust to new environmental conditions, complementing what was observed by Bora et al. (2020). Although the invasion status of this species in the middle and lower estuary area is low, our results show the possibility of expansion of colonization in areas colonized by native species. These areas are very critical for conservation and, therefore, more complex studies are needed to better estimate the invasiveness of U. arrecta in saline ecosystems and to promote control or management policies for this species in these areas.