Alternanthera philoxeroides (Mart.) Griseb. (commonly called alligator weed) is an amphibious, perennial herb of the Amaranthaceae family, native to South America (Holm et al. 1997; Julien et al. 2012). The species is listed as one of the most invasive species in China (Li and Xie, 2002). Populations of A. philoxeroides in China have extremely low genetic diversity and rarely produce fertile seeds within an entire life cycle (Xu et al. 2003; Ye et al. 2003). Thus, the species mainly achieve offspring recruitment via vegetative means such as stolon and root fragments (Jia et al. 2009; Dong et al. 2010, 2012). Clones of A. philoxeroides can establish extensive networks of connected ramets, and clonal integration can remarkably promote the individual performance and the population expansion of A. philoxeroides (Wang et al. 2008; Yu et al. 2009; Xu et al. 2010; Dong et al. 2015; Xi et al. 2019). A. philoxeroides has spread widely in both aquatic and terrestrial habitats, such as irrigation ditches and riparian crop fields, causing severe ecological and environmental problems (Pan et al. 2006; Wu et al. 2016).
On December 9, 2016, 150 stolon fragments of A. philoxeroides were collected from three separate populations (approx. >500 m apart) in a riparian agricultural area (28.87°N, 121.01°E) in Taizhou City in Zhejiang Province, China. These stolon fragments were then transported to Beijing Forestry University in Beijing on December 10, 2016. On December 11, 2016, 30 similar-sized clonal fragments were randomly selected for the experiment and classified into two parts. One was defined as the “apical part” consisting of one main stem and one lateral branch, and the other as the “basal part” consisting of two relatively older lateral branches. The main stem and three branches each had three nodes (Fig. 1).
The experiment employed a two-way factorial design, with N level treatments (i.e., 40 or 120 mg N L− 1; Fig. 1) crossed with the position of 15N supply treatments (i.e., 15NO3+ supplied in the apical part or the basal part; Fig. 1a, b, d, and e). There were five replicates for each of four combined treatments. To explicitly measure the concentration of 15N derived from 15N-labelled nitrate, five additional replicates of clonal fragments were used as a control treatment for each of the N levels (i.e., external 15NO3+ supplied in neither the apical part nor the basal part; Fig. 1c and f). Each of the clonal fragments was placed into a pair of adjacent plastic cups (with a 1000 mL capacity) with the apical part of the fragment in one cup, the basal part in the other, and the internode that connected the two parts running through matching notches in the rims of the cups. In the N level treatments, clonal fragments were grown in modified Hoagland solutions containing either 40 or 120 mg N L− 1, supplied as Ca(NO3)2, with 15 clonal fragments grown in each solution. We varied the concentration of CaSO4 between solutions to maintain the same total solute concentration and the same concentration of each nutrient except SO4 − 2 in each solution (Alpert et al. 2002; Wang et al. 2017). The modified Hoagland solution was refreshed every five days.
To test the acropetal transport of N between connected ramets, the basal parts of five clonal fragments in each of the N level treatments were labelled by Ca(15NO3)2 (99.24 atom%; Shanghai Research Institute of Chemical Industry, Shanghai, China), one day before the harvest. To test the basipetal transport of N, the apical parts of another five clonal fragments in each of the N level treatments were similarly labelled with Ca(15NO3)2. We used Hoagland solutions containing 15N, where the amount of 15N from Ca(15NO3)2 occupied 10% of the total amount of N in the solution. The plants receiving 15N supply treatments were allowed to take up 15N for 25 h and then were harvested. The plants in the control treatment (i.e., the remaining five clonal fragments in each N treatment) were not labelled by Ca(15NO3)2 but were harvested at the same time.
The experiment was conducted at the Wetland Process Lab in the School of Nature Conservation, Beijing Forestry University, and it lasted for five weeks from December 12, 2016, to January 15, 2017. The mean room temperature during the experiment was 23.34 ± 0.25°C. The light source was supplied by full-spectrum LED lamps (Guangdong Shunde POVI Biological Technology Co., Ltd., Foshan, Guangdong) for 12 h of light per day. The irradiation level of lamps was kept at an average of 95 µmol m− 1 s− 1.
Measurement and isotope analysis
At harvest, leaves, stems, and roots of the apical part and the basal part of clonal fragments were dried at 70°C for 48 h, weighed to measure biomass, and ground using a Retsch MM400 Mixer Mill at a frequency of 28 Hz for 6 min (Retsch GmbH, Haan, Germany). A subsample of 2 mg powder was used to measure the N concentration and the atom% 15N of leaves, stems, and roots, using a Flash 2000 Elemental Analyzer that was interfaced with a Delta V Isotope Ratio Mass Spectrometer (Thermo Fisher Scientific, Inc., USA).
In each of the N level treatments, atom% excess 15N (APE) was calculated by the atom% 15N difference between plants in each of the N supply treatments (atom% 15Ntreatment) and the ones in the control treatment (atom% 15Ncontrol), i.e., APE = atom% 15Ntreatment - atom% 15Ncontrol (Gao et al. 2014; He et al. 2009). The 15N concentration of each plant organ (mg 15N g− 1 d.w. plant organ) was calculated by multiplying the N content (mg N g− 1 d.w. plant organ) and APE. The amount of 15N in each plant organ (mg) was then calculated by multiplying the 15N concentration of the plant organ and the mass of the corresponding plant organ. The amount of 15N in the apical or basal part (mg) was calculated according to the sum of the amount of 15N in the leaves, stems, and roots of the apical or basal part; thus, the 15N concentration of the apical or basal part (%) was calculated by dividing the total 15N amount of the apical or the basal part by the mass of the corresponding part.
The partitioning proportion of 15N among organs (in either the apical part or basal part of clonal fragments) was calculated by dividing the amount of 15N in the plant organ by the total amount of 15N in the corresponding part (e.g., the apical or basal part). The rate of 15N transport toward recipient ramets was calculated by dividing the amount of 15N in recipient ramets by the total amount of 15N in the whole clonal fragment.
Two-way ANOVAs were used to test effects of the position of 15N supply (apical part vs. basal part) and N levels (40 vs. 120 mg N L− 1) on the amount of leaf 15N, stem 15N, root 15N, and the total amount of 15N in the apical and basal parts of the clonal fragments of A. philoxeroides. Two-way ANOVAs were also used to test effects of the position of 15N supply (apical part vs. basal part) and N levels (40 vs. 120 mg N L− 1) on the partitioning proportion of leaf 15N, stem 15N, and root 15N of the apical or basal parts of clonal fragments. In addition, linear regressions were employed to examine the correlation between the transport rate of 15N toward recipient parts (transport rate of 15N = the amount of 15N in the recipient part/the total amount of 15N in the clonal fragment) and ΔPNC (ΔPNC = the N concentration of the apical part - the N concentration of the basal part). Linear regressions were also employed to examine the correlation between the partitioning proportion of 15N of each plant organ (the amount of 15N in the plant organ/the total 15N in the apical or basal part) and PNC (the N concentration of the apical or basal part) of the apical part or the basal part of clonal fragments. Data that violated the assumptions of homogeneity of variance and normality were natural-log transformed. Data analyses were conducted using R v.4.1.1 (R Core Team 2021).