We employed a chronosequence approach (space-for-time trade-off) in which, more than 200 live overwintered P. crassipes ramets were collected from different sampling locations (Table 1) and cultivated under three water levels to explore the effects of water level variation on plant growth in spring. The chronosequence approach, in which spatial differences are replaced by temporal changes, was adopted to study the effect of water level variation on plant growth and to determine the unique survival and phenotypic plasticity strategies of plant roots. Instead of waiting for global warming changes to occur over time, we selected different sampling locations that represent different stages of this process. Notably, January is the coldest month across the three sampling locations.

Pontederia crassipes ramets subjected to freezing injury were collected from a bay (31.0338°N, 120.4228°E) outside the Taihu Laboratory for Lake Ecosystem Research (TLLER), Dongshan Branch (DB), in the Yangtze drainage basin on February 22, 2022. The area exhibits a mean January average air temperature of 3.0°C, and the minimum winter temperature (-6°C) occurred on December 26 from 2021–2022 (https://lishi.tianqi.com/suzhou). As a region characterized by low winter temperatures, Suzhou contains the northernmost distribution of P. crassipes. The littoral zones in this area, such as those around Taihu Lake, offer nutrient-rich but freezing-prone environments. This setting allows the study of the mechanism through which the plant rooting strategy mitigates freezing damage in mud during winter drawdown periods.

Pontederia crassipes ramets subjected to chilling injury were collected from a riverbank in Nanping city, Fujian Province (26.6022°N, 118.1457°E), in the Minjiang drainage basin on February 25, 2022. The minimum winter temperature (1°C) occurred on February 21, 2022 (https://lishi.tianqi.com/nanping). The distance between Nanping and Suzhou is 540 km. Situated in Fujian Province, Nanping exhibits moderate winter temperatures. This area represents regions where chilling injury (not freezing injury) is the dominant limiting factor for invasive aquatic plants. The selection of Nanping enables the evaluation of the adaptability of the plant under intermediate climatic conditions, thus reflecting environments where climate change could shift temperatures toward higher, less restrictive levels.

Pontederia crassipes ramets subjected to a warm location, i.e., neither a freezing nor chilling location, were collected on February 26, 2022, in Guangzhou, Guangdong Province (23.3061°N, 113.2773°E), in the Pearl River drainage basin. The minimum winter temperature (4°C) occurred on February 20, 2022 (https://lishi.tianqi.com/guangzhou). The distance between Guangzhou and Nanping is 612 km. Guangzhou represents an environment where P. crassipes thrives without temperature-induced stress. The inclusion of this location allows for the comparison of plant traits under optimal growth conditions, thus serving as a baseline for assessing the impact of colder climates.

The mesocosm experiment was conducted at the TLLER, DB (31.0331°N, 120.4217°E), which is located on the shore of Taihu Lake. This area enjoys a mild subtropical climate.

Prior to the experiment, young ramets obtained from the three sampling locations were cultivated separately in a greenhouse in the TLLER, DB. The experiment started on March 13, 2022. Sediment was collected from Taihu Lake near the TLLER, DB. A total of 15 cm of sediment was added to each cylinder (d = 0.30 m, h = 1.00 m). All the cylinders were placed on a platform in a natural setting in the TLLER, DB. No protection measures (for instance, shading and pest control) were implemented. Young ramets of a similar shape and weight were collected, and the biomass was measured. The water level was set to 1, 10 and 20 cm. These water levels were selected on the basis of previous field investigations and experimental evidence (Huang et al. 2022; Wang et al. 2017). The 1-cm depth represents stranded plants in moist littoral zones, highlighting stress conditions during drawdown. The 10-cm depth represents the optimal rooting conditions observed in littoral environments, which enhances nutrient uptake and survival. Finally, the 20-cm depth represents free-floating conditions, which is relevant for understanding the adaptability and dispersion of this plant under water level rise. These levels are consistent with the natural conditions in the Lake Taihu Basin, where seasonal fluctuations significantly influence the growth and spread of P. crassipes. Each treatment was replicated six times (n = 6). The water level was maintained with a pump after rainfall.

The experiment ended on May 20, 2022, when the weather neared summer conditions. The sediment near the roots was removed along the direction of the roots, and the adhering sediment was gently washed away by a pump during harvesting. The number of diaspores and the lateral root number c of the ramets in each cylinder were counted. The original root shape was maintained to the greatest extent possible. The root system was cut with tweezers so that the roots were dispersed and did not overlap.

The plant functional traits included the total biomass, shoot/root (S/R) ratio, diaspore number, maximum quantum yield of photosystem II F_v/F_m, leaf area (LA), specific leaf area (SLA), root length (RL), specific root length (SRL), lateral root number c, mean rootlet number a, and topological indices TI, q_a and q_b (Table 2).

On the harvest day, five randomly chosen leaves were sampled from the plants in each treatment with a 4-mm (0.13-cm²) diameter leaf clip. The leaves were incubated in the dark for 20 minutes before sunrise to ensure that all the reaction centers within the chloroplasts were fully oxidized, which was assessed via a handheld plant efficiency analyzer (PEA) (Hansatech Instruments, Ltd., Norfolk, UK). The initial fluorescence F₀ and maximum fluorescence F_m of the leaves were measured, and the maximum quantum yield or energy trapping efficiency of the photosystem II reaction centers, F_v/F_m, was calculated as follows:

F_v/F_m = (F_m - F₀)/F_m.

The biomasses of the plant shoots and roots were determined via weighing after drying at 60°C for 48 h in an oven.

Images of the plant leaves and roots were developed by scanning via an Epson 12000XL scanner (Seiko Epson Corp., Suwa, Nagano, Japan). Following the recommendations of Bouma et al. (2000), the image resolution was set to 600 dpi, and the scanned images were saved as 1:1 uncompressed 8-bit high-resolution TIFF files. To increase the contrast between the roots and the background, the nearly transparent roots of P. crassipes were stained with a 0.2 mmol/L (0.075 mg/mL) methylene blue solution for 30 s before scanning with a white LED light source. The scanned root images were processed via WinRHIZO root measurement and analysis software (Regent Instruments Inc., Québec City, Canada). WinRHIZO software was chosen for its ability to provide detailed quantitative data on root morphology and topology, such as the LA, RL, and rootlet number a to better elucidate the adaptive strategies of P. crassipes.

The plant SLA was calculated as follows:

SLA = LA/leaf biomass.

The SRL was calculated as follows:

SRL = RL/root biomass.

The root system of P. crassipes is highly complex and extensively overlapped, making it impossible to obtain a complete image via conventional scanning analysis methods. In this study, we investigated the overall root structure by cutting and individually scanning roots and then combining them with the original root branching.

The root topological indices TI, q_a and q_b were obtained according to our previously published methods (Huang et al. 2019):

$\mathrm{TI}=\frac{\log a}{\log ca}$;

$q_a=\frac{a-1-\ln ca/\ln 2}{ca-1-\ln ca/\ln 2}$;

$q_b=\frac{\left(a+3-2/a\right)/2-1-\ln ca/\ln 2}{\left(ca+1\right)/2-1/ca-\ln ca/\ln 2}$.

The topological indices TI, q_a and q_b of P. crassipes are indicated by the lateral root number c and the mean rootlet number a.

For herringbone-like branching, the value of the topological index TI is close to 1, with the values of both q_a and q_b approaching 1. In contrast, dichotomous branching yields a TI value close to 0.5, with q_a and q_b values close to 0. According to plant root structure theory, plants with herringbone-like branching are adapted to low-nutrient environments because this branching pattern features more and longer connections and a lower root system hierarchy. However, this branching pattern exhibits a lower transmission efficiency and higher construction cost. Dichotomous branching root systems encompass shorter connections and are easier to construct and maintain than are herringbone-like root systems. This root branching pattern typically occurs in environments in which nutrients and water are more abundant.

Before analysis, all the data were analyzed to ensure that they met the assumption of a normal distribution on the basis of the Shapiro‒Wilk test and the assumption of homogeneity of variance on the basis of the Levene test. If the data did not satisfy these assumptions, they were transformed with a log(x+1) function. An unconstrained unimodal ordination model based on detrended correspondence analysis (DCA) was constructed in Canoco 5.0 (Microcomputer Power, Ithaca, NY, USA). In this study, the results revealed that the lengths of the gradients were shorter than 3, and therefore, the use of linear principal components analysis (PCA) was preferred. A general linear model (GLM) was employed to examine the effects of the sampling location (SL) and water level (WL) on the plant functional traits. Moreover, the effects of the interaction between SL and WL on plant functional traits were assessed. Post hoc pairwise comparisons of the means were conducted via Duncan’s multiple range test at a significance level of P = 0.05 to test for differences between treatments. One-way analysis of variance (ANOVA) was employed to test for differences in plant functional traits at a significance level of P = 0.05. All statistical analyses were performed via SPSS Statistics 26 (IBM Corp., Armonk, NY, USA).

Unlike Liu et al. (2016), who reported significant impacts of the temperature, our findings highlight the water level as a critical factor influencing plant traits. The littoral zone contains abundant nutrients, and the temperature is higher than the air temperature, which can help plants overwinter, survive and proliferate during the vernal flooding period. The shift in P. crassipes from being rooted to free-floating driven by water level may be regarded as a unique survival strategy based on phenotypic plasticity. Other free-floating plants with similar rooting behavior have not been reported to overwinter in China. Thus, the shift in the rooting behavior of P. crassipes may indirectly facilitate it to suppress other species in spring. This furthers the plant invade empty niches left on the water surface after freezing-induced mortality of other free-floating plants; this agrees with the empty niche hypothesis, which states that invasive plants exploit new habitats created by fluctuating resources (Hussner et al. 2017; Golivets and Wallin 2018; Mathakutha et al. 2019). These findings suggest that P. crassipes exhibits significant plasticity in response to water level changes, which can be considered a crucial survival strategy in dynamic aquatic environments. This plasticity may enable P. crassipes to colonize new areas, contributing to invasion success.

The ability of P. crassipes to survive at low temperatures by rooting in littoral zones suggests that it can withstand and adapt to colder climates more effectively than previously thought. The 10-cm water depth likely provides optimal conditions for the overwintering of P. crassipes by balancing accessibility to water and nutrients while minimizing environmental stress. At this moderate depth, its rooting structures can securely anchor in nutrient-rich littoral sediments, thus supporting growth and biomass accumulation. This depth also protects the roots and stolons against temperature extremes, insulating them within the stable thermal environment of the littoral zone—an advantage during overwintering. Its ability to develop functional traits such as an increased biomass, diaspore number, and root length at this depth highlights its adaptive strategies to survive and proliferate under changing environmental conditions, such as vernal water rise. As global warming continues, the minimum winter temperatures are projected to increase, potentially removing the historical thermal barriers limiting the northward expansion of invasive species. This could lead to expansion of their geographical range, allowing P. crassipes to invade new water bodies that were previously unsuitable due to low winter temperatures.

Given the potential for P. crassipes to expand its range under climate change, early detection and rapid response strategies are crucial. Monitoring programs should be established in vulnerable areas, such as littoral zones, to detect new invasions early and take prompt action to control their spread before the plant becomes established. By identifying the water level as a critical factor influencing the growth of P. crassipes, we provide new insights into the mechanisms that facilitate its invasion and persistence in freshwater ecosystems. This understanding is essential for predicting the spread of P. crassipes under future climate scenarios in which hydrological patterns may change. For example, regulating water levels in winter could be a potential method to limit the growth and spread of this invasive species.

The terrestrial plant Arabidopsis thaliana has been widely studied as a model plant across almost all plant subdisciplines (Pang and Meyerowitz 1987; Meinke 1998; the Arabidopsis Genome Initiative 2000; Poecke and Dicke 2008). Among aquatic plants, Phragmites australis has been employed as a model organism for environmental changes, highlighting its utility in research on global change (Holdredge et al. 2010; Lambertini et al. 2012; Meyerson et al. 2016; Eller et al. 2017). Most studies on P. australis have focused on its ability as a wetland plant to tolerate different water and nutrient conditions but not on specific seasonal rooting behaviors, as observed in P. crassipes (Lambertini et al. 2012). The rooting behavior of P. crassipes in littoral zones in winter, which allows it to withstand low temperatures and proliferate in spring, is relatively unique. Several models of the growth and yield (Lorber et al. 1984), population dynamics (Gamage and Asaeda 2004), recognition based on airborne hyperspectral imagery (Yang and Everitt 2010), natural resource valuation (Buller et al. 2013), growth and interactions between shoot and root systems (Eid and Shaltout 2017) and root topology (Huang et al. 2019) of P. crassipes have been established. Numerous studies on the role of this species in element cycling in freshwater ecosystems (Pinto-Coelho and Barcelos Greco 1999); impact, management and utilization (Malik 2007; Villamagna and Murphy 2010; Coetzee and Hill 2012); relationships with herbivores (Fan et al. 2016); biosorption of chemical pollutants (Mohanty et al. 2006; Alvarado et al. 2008; Malar et al. 2016); response to environmental factors (You et al. 2013; Fan et al. 2015; Wang et al. 2017); and phytoremediation (Feng et al. 2017; Mishra and Maiti 2017) have been published as case studies. The relatively large and abundant leaves of this species make it easier to estimate its foliar traits more precisely, and its root system is relatively easy to quantify. We recommend P. crassipes as a model organism for studying the responses of invasive aquatic plants to global changes in freshwater ecosystems because this species is a successful invader. Thus, its use could be beneficial for identifying operative methods to control its spread.