Branches of M. aquaticum were harvested in January, 2019, at Baoan Lake, Hubei Province (30°15'51.92"N, 114°43'12.33"E). Subsequently, they were submerged in an aquarium filled with water for pre-cultivation. Two days later, healthy uniformly sized shoots of M. aquaticum were selected for transplantation.

This study was conducted in January, 2019, in a greenhouse at the Hubei Normal University Aquatic Environment and Ecology Restoration Laboratory. Four hundred and eighty M. aquaticum plants, pre-cultivated and uniformly grown, were chosen and weighed to determine their initial weight. Each plant’s average fresh weight was 2.47 ± 0.05 g, with an average height of 25.39 ± 0.62 cm. Sixty plants were air-dried to measure their initial dry weight, which was used to calculate moisture content. The remaining 420 plants were planted in experimental buckets, each containing 20 plants. The buckets had a height of 56 cm, an upper diameter of 52 cm, a lower diameter of 38 cm, and a volume of 90 L. Next, a 12 cm layer of sediment was applied to the pots, which originated from the eutrophic Qingshan Lake. The sediment contained 1,201.77 mg/kg of total phosphorus (TP), 620.74 mg/kg of total nitrogen (TN), and 71.32 mg/kg of organic matter (OM).

TN concentrations in the lake substrates were analyzed using the semi-micro Kjeldahl method following digestion with H₂SO₄ and HClO₄, as indicated by Wang et al. (2017). TP concentrations were determined using the ascorbic acid method, as suggested by Varol and Sen (2012). OM concentrations were determined using the potassium dichromate-sulfuric acid method, as described by Zhang et al. (2015).

The water level in each container was continuously maintained as the M. aquaticum plants were inundated. The water used was extracted from the Qingshan Lake (TN: 3.23 mg/L, TP: 0.32 mg/L). Seven water temperatures (0 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, and 30 °C) were selected for the experiment, with three duplicates for each temperature. A thermometer recorded the lake water temperature at 5 °C. Ice cubes were added to the buckets of the 0 °C treatment group to maintain the experimental temperature. A heating rod, equipped with four probes, was inserted into the water in the middle of all treatment groups except those kept at 10 °C to provide heat. On April 1, 2019, the experimental plants were harvested after a 72-day experimental period.

Growth indicators

The height, number of stems diameter, and number of stem nodes were measured every 3‒7 d, whereas the length, diameter, and number of branches of each stem were measured using Vernier calipers and a tape-measure every 10‒12 d. Nine M. aquaticum plants were randomly selected from each temperature treatment bucket to determine their average values. The biomass of M. aquaticum was weighed at the end of the experiment at each temperature to obtain the total weight.

Physiological indicators

After harvesting, three–five young and undamaged leaves were selected from the top of the main stem. To determine chlorophyll content, 0.2 g of fresh leaves was weighed and subjected to the 80% acetone extraction method (wavelength: 665, 649, and 470 nm) under light-avoidance conditions by placing the samples in the dark (Wang 2006).

Fresh leaves (0.5 g) were weighed to determine activity levels of catalase (CAT), an antioxidant enzyme that is widely found in organisms (Hu et al. 2016). CAT activity was assessed spectrophotometrically by monitoring the reduction in hydrogen peroxide (H₂O₂) absorbance at 240 nm based on the method established by Beers and Sizer (1952). Data were recorded at 1-min intervals for 4 min. The magnitude of CAT activity was denoted as the amount of H₂O₂ decomposed over a specific timescale, as explained by Saxena et al. (2016) and Wang et al. (2017). The initial minutes of the reaction exhibited a stronger response, which gradually subsided as the enzyme was depleted. CAT contributes to the development of heat-induced cold tolerance and its activity is positively correlated with increased cold resilience (Sala and Lafuente 1999).

For the test conducted to grow M. aquaticum, water indicators were determined using the following method: TN concentrations in the experimental water were determined using the alkaline potassium persulfate oxidation-UV photometric method (wavelengths: 220 and 275 nm), whereas TP was determined using the potassium persulfate digestion method (wavelength: 700 nm) (Zhu et al. 2022).

Microsoft Excel 2003 (Microsoft Inc., Redmond, WA, USA) was used for data processing, and correlation and analysis of variance were performed using SPSS 27.0.1 software (SPSS Inc., Chicago, IL, USA). To examine differences in biomass, leaf pigment content, and CAT activity at different temperatures, one-way analysis of variance (ANOVA) was performed. Data were assessed for normality and homogeneity (P > 0.05) using the Shapiro–Wilk and Levene methods, respectively. Transformations were performed on data that did not meet the assumptions of normality or variance homogeneity, followed by post hoc testing using the Waller-Duncan method. Growth index analysis of M. aquaticum was conducted using Origin 8.5 software (Origin Labs Inc., Northampton, MA, USA).

Over time, M. aquaticum grew rapidly during the first 20 days, but slowed from days 20 to 50 and continued to grow from days 50 to 75 (Figure 1). Temperature had a noticeable influence on plant height. In all the treatment groups, an increase in water temperature resulted in an increase in plant height. The tallest plants, measuring 93 cm, were observed on day 72 at 25 °C. The plant height increased gradually under treatments of 0 °C and 5 °C, registering the highest values of 38.97 cm and 37.98 cm, respectively, on day 72.

Figure 1. 

Height of Myriophyllum aquaticum at different water temperatures.

Water temperature affected stem diameter, as shown in Figure 2. Stem diameters were greater in treatment groups at 5 °C, 25 °C, and 30 °C. Between days 0 and 67, stem diameter peaked under the 5 °C treatment. On day 39, stem diameter grew more rapidly in the 25 °C and 30 °C treatment groups, surpassing those at 5 °C that reached a peak of 1.35 cm on day 67. By day 72, stem diameter was maximized at 1.67 cm under the 25 °C treatment.

Figure 2. 

The stem diameter of Myriophyllum aquaticum at different water temperatures.

Differences in the number of stem nodes in M. aquaticum were observed at different water temperatures (Figure 3). Initially, each plant contained an average of 10 ± 1 stem nodes, but noticeable variation emerged after 15 days. After day 15, the maximum quantity of stem nodes, reaching 25 ± 3 per plant, was observed at both 25 °C and 30 °C, whereas at 0 °C and 5 °C, only 13 stem nodes were observed per plant. By day 72, the group subjected to a 25 °C treatment had the highest number of stem nodes, with 51 per plant. The amount of stem nodes in the groups treated at 0 °C and 5 °C remained relatively stable from day 20 onwards, accounting for 30% of the stem node number of the 25 °C group.

Figure 3. 

Number of stem nodes of Myriophyllum aquaticum at different water temperatures.

Except for 30 °C, the number of branches increased progressively with growth of the plants at different water temperatures. The numbers of branches at 0 °C, 5 °C, and 10 °C were lower than that at the other water temperatures (Figure 4). Differences in the number of branches were observed starting on day 24. Subsequently, by day 34, the number of branches had declined at 30 °C, gradually leading to their disappearance. At the termination of the experiment, the greatest amount of branches, 134 per plant, was noted at 15 °C.

Figure 4. 

Number of branches and average branch length of Myriophyllum aquaticum at different water temperatures.

Total biomass initially increased and subsequently decreased with rising water temperatures, with observed differences in biomass among the various water temperature treatments (Figure 5). Significantly higher biomass was found in the 20 °C treatment group compared to the 0 °C, 5 °C (P < 0.01) and 30 °C (P < 0.05) treatment groups.

Figure 5. 

Total biomass of Myriophyllum aquaticum under different water temperatures.

Effects of temperature on pigment content of M. aquaticum

The levels of carotenoid (Car), chlorophyll a (Chl-a), chlorophyll b (Chl-b), and total chlorophyll (total Chl) were the lowest at 20 °C. The Car, Chl-a, and total Chl contents were highest at 10 °C, whereas the Chl-b content was highest at 15 °C, as shown in Figure 6. However, the fluctuations in Chl-a, Chl-b, and total Chl contents were less prominent than those of Car at different temperatures. Considering all temperatures, Chl-a and Chl-b ranged from 0.66‒0.92 and 0.14–0.20 mg/g, respectively, throughout the experiment. Moreover, Car and total Chl content were 0.16 to 0.27 and 0.80 to 1.09 mg/g, respectively.

Figure 6. 

Pigment content in the leaves of Myriophyllum aquaticum at different water temperatures.

Effect of temperature on CAT activity in M. aquaticum

Reaction time and CAT activity were directly proportional to the amount of CAT in equal-quality leaf fractions. When the amount of CAT was high, the H₂O₂ decomposition rate was higher than that when the amount of CAT was low. In the first minute of the reaction, CAT activity was highest at 5 °C (51.56 g/min), followed by 20 °C (29.38 g/min), and lowest at 15 °C (17.50 g/min). CAT activity in the second minute of the reaction was similar. During the final two minutes of the reaction, the activity of CAT was highest at 5 °C, closely followed by 30 °C. Treatment at 5 °C had the most significant effect on the activity of M. aquaticum CAT (Figure 7).

Figure 7. 

Catalase activity of Myriophyllum aquaticum under different water temperatures.

This study showed that M. aquaticum growth and development were significantly affected by different water temperatures. Height, biomass, and growth rates were higher at 10‒25 °C. Conversely, high water temperatures (30 °C) inhibited the plant, resulting in lower height and number of stem nodes. On day 34, the number of stem nodes was significantly decreased at 30 °C, whereas it increased at 0–25 °C. The overall biomass of M. aquaticum at 30 °C was lower than that at 10–25 °C, as the plants had fewer and shorter branches. High water temperatures led plants to expend more energy for metabolism, thereby reducing the number of branches (Wang et al. 2008). Therefore, 10–25 °C had a positive effect on the growth of M. aquaticum, whereas high water temperatures (30 °C) were not suitable.

In our study, low-water temperatures (0–5 °C) hindered the growth of M. aquaticum (excluding stem diameter). Cold stress induces several physiological and biochemical changes in plants in response to the damage caused by low temperatures through self-regulation (Quan et al. 2023). Thus, M. aquaticum responds to low-temperature stress by decreasing plant height and increasing stem diameter. Specifically, at 5 °C, M. aquaticum exhibited the shortest plant height but the greatest stem diameter compared to the other temperatures throughout the experiment until day 67. The advantages of dwarf plants, such as maintaining optimal temperatures and increasing heat absorption while reducing heat loss, have been previously demonstrated (Jiang et al. 2020).

The adaptation of M. aquaticum to low-temperature stress was demonstrated by the observed changes in CAT activity. CAT activity increased with decreasing temperature, indicating that low temperatures can induce an increase in CAT activity to mitigate damage (Wang et al. 2019). However, with increasing stress, the antioxidant defense system can be damaged and enzyme activity diminished. Wang et al. (2019) provided further information concerning this topic. In this study, it was found that M. aquaticum exhibited higher CAT activity when subjected to 5 °C low-temperature treatment as compared to the 10–30 °C treatment (P < 0.01). This suggests that M. aquaticum responds to low-temperature stress by increasing its CAT activity, a phenomenon also reported by Zhang et al. (2013). Therefore, we can infer a direct correlation between elevated cold tolerance and CAT activity. One explanation for the reduced CAT activity at 30 °C is the insensitivity of M. aquaticum CAT towards high temperatures. High-temperature stress in M. aquaticum can produce significant amounts of reactive oxygen species (ROS) that trigger lipid peroxidation of membranes, inhibit enzyme expression, disrupt enzyme-active centers, or change enzyme structures, resulting in decreased CAT activity (Zhou et al. 2010). CAT increases photosynthetic pigment content in plants (Nan and Fan 2008). In this experiment, CAT content was highest at 5 °C. However, the photosynthetic pigments content did not reach their maximum. This discrepancy could potentially be attributed to prolonged exposure to low temperatures causing detrimental effects to the chloroplast structure and subsequently impairing both the functionality and efficiency of photosynthetic organs within the chloroplasts (Fang et al. 2023).

M. aquaticum, a tropical species native to South America, thrives in warm, humid, and sunny climates, and is widely distributed in tropical and subtropical regions (Liu et al. 2017). Heat and water temperature are the leading environmental factors that influence the growth and reproduction of M. aquaticum.

In Huangshi City, the average daily maximum temperatures during 2018 were 22 °C in spring, 32 °C in summer, 23 °C in fall, and 10 °C in winter (China Weather 2018). In our study, the temperature treatments were set to simulate the different seasons: 10–25 °C during spring and fall, 30 °C in the summer, and 0–10 °C in the winter. At both 20 °C and 25 °C, M. aquaticum survived and produced the highest number of new shoots. This suggests that the temperature for the growth of this species is between 20 °C and 25 °C (Ditomaso and Healy 2003). Especially, at 10–25 °C, M. aquaticum experienced an increase in biomass and leaf photosynthetic capacity. The increased capacity for leaf photosynthesis was evident from the increase in Chl content.

During winter (0–10 °C), the dormancy of M. aquaticum contributes to its competitiveness in various environments. M. aquaticum undergoes partial winter dormancy, with only the upper aerial portion decaying. During this time, the plant overwinters with its roots submerged in the mud. As the water temperature increases in spring, the upper stems regenerate. There were no instances of disease or death throughout the growth period, suggesting a robust capacity for adaptation to the environment or regeneration (Ditomaso and Healy 2003; Liu et al. 2017).

In the Northern hemisphere, macrophytes rouse from winter and re-start producing biomass during spring with an increase in both air and water temperatures, as well as longer day length (Silveira and Thiebaut 2017). Common submerged plants in China, such as Vallisneria natans (V. natans), H. verticillata, Potamogeton maackianus (P. maackianus) and P. crispus, differ in their life histories from M. aquaticum. V. natans and H. verticillata overwinter with winter buds, then seeds and sprouts in the spring. However, germination from seed or winter buds must to meet certain conditions (nutrients, temperature, etc.) (Nielsen et al. 2003; Xiao et al. 2010) and time, and the survival rate is easily affected by environmental factors during seedling growth. Although P. maackianus and P. crispus begin sprouting in early April, they reach their maximum biomass in July and then gradually wilt and die (Wang et al. 2015). However, M. aquaticum begins to grow in spring but does not die-off in summer and fall. During fall, M. aquaticum peaked. M. aquaticum can also grow during the over winter. In winter, we observed that even when M. aquaticum was frozen, it did not die and could still grow even after the ice had melted. In our experiments, even if the temperature was only 0‒5 °C, the M. aquaticum could still grow. Although its growth was relatively slow, until the end of the experiment, M. aquaticum did not die under conditions of 0‒5 °C. This is attributed to its reliance on roots for winter survival (Liu et al. 2017). In addition to seed reproduction, submerged plants can also reproduce asexually using stolons, rhizomes, bulbs, tubers and fragments (Titus and Hoover 1991; Riis and Sand-Jensen 2006). After asexual reproduction, a certain amount of time is required to establish roots and then colonize and grow after successful rooting. Moreover, such plants are equally susceptible to environmental conditions such as seed germination. In spring, because M. aquaticum retains its original roots and stems before dormancy, it can continue growing. Compared to other plants, M. aquaticum does not need to spend time on seed germination or root formation. This allows M. aquaticum to occupy its ecological niches relatively quickly.

Based on the growth and physiological indicators of M. aquaticum at various temperatures, the ideal water temperature range for its growth is between 20 °C and 25 °C. However, M. aquaticum can regulate itself under high and low-temperature stress to survive and thrive at temperatures ranging from at least 0 °C to 30 °C. This adaptation is advantageous for M. aquaticum when invading subtropical and temperate regions, where temperature variations occur throughout the four seasons.