Effects of Waterlogging Stress on the Neighboring Relationships between Cleistocalyx operculatus (Roxb.) Merr. and Dalbergia odorifera T. Chen Saplings

Abstract

Neighboring relationships among plants have been extensively reported, but little is known about the effect of waterlogging. In this study, Cleistocalyx operculatus (Roxb.) Merr. and Dalbergia odorifera T. Chen saplings were used in investigating the neighboring relationships between legumes and non-leguminous woody plants under different planting and watering regimes. Results showed that C. operculatus and D. odorifera are waterlogging-tolerant species, and C. operculatus with high proportion of adventitious roots would be at an advantage during waterlogging. The growth performance of D. odorifera was better than that of C. operculatus under well-watered single-planting conditions. However, under well-watered mixed-planting conditions, C. operculatus had an obvious inhibitory effect on the growth traits (increments in stem height and leaf number, total leaf area, and total plant fresh weight) and physiological responses (net photosynthesis rate, stomatal conductance, and transpiration) of D. odorifera, whereas the growth of C. operculatus slightly increased in the presence of D. odorifera. Under waterlogging mixed-planting conditions, the facilitative effect was more intensive; the total leaf area, underground fresh weight, and total plant fresh weight of C. operculatus significantly increased, but a negative effect was found in D. odorifer. These results showed that the neighboring relationship between these two species is predominantly favorable to C. operculatus. This research sheds new light on screening waterlogging-tolerant arbor species and species collocation during vegetation restoration and reconstruction activities in wetland systems.

1. Introduction

Plant–plant interactions are fundamental factors in mediating plant population dynamics, plant community structures, and ecosystem functions [1,2,3]. Ecologists have focused on competition as the most important interaction among neighboring plants [4]. It is widely acknowledged that plants must compete for always-limited resources (e.g., water, nutrients, light, or space) and try to overthrow each other when in proximity [5,6,7]. More recently, accumulating evidence has shown that facilitation may play a greater role in plant–plant interactions than competition [8,9,10,11]. In some cases, they can be very specific, i.e., the mixed culture of legumes and nonleguminous plants can promote the N uptake of non-leguminous plants [12], and facilitative interactions are vital to the growth, reproduction, and survival of neighboring plants [13,14]. However, interactions among plants are not always constant. Several studies have reported that the direction and intensity of plant–plant interactions can be altered by abiotic factors, neighbor characteristics, and target plant traits [15,16]. Thus, an in-depth understanding of how interactions among plants change with external abiotic conditions and neighboring plants is essential.

As one of the most prevalent types of abiotic stress, waterlogging limits the growth and reduces the survival of many terrestrial plants, especially in tropical and subtropical areas [17]. Over 17 million km² of land surface on Earth is affected by waterlogging. This stress is expected to increase in the future owing to the projected tendency of precipitation to intensify and to increase in rainfall as a result of global climate change [18,19]. Waterlogging causes severe changes in cell physiology, energy consumption, and growth of the plants [20]. Insufficient O₂ supply in the roots is a major growth-limiting factor for plants and restricts energy generation, affecting the water supply for cell expansion and leaf growth, and exerting adverse effects on photosynthetic rate [21]. Moreover, extra water in waterlogged soil can induce oxidative stress and stomatal closure, reduce chlorophyll content, and ultimately limit light interception in plants [22]. Plants have evolved two types of strategies, escape strategy and quiescence strategy, to cope with waterlogging-induced hypoxia [23]. However, the adaptation strategies of plants to waterlogging stress may affect the balance of plant–plant interactions. Da Silva et al. [24] reported that one likely mechanism of facilitation in flood tolerance is the amelioration of anoxic rhizosphere environments by oxygen leakage from neighboring plants. This finding is consistent with the finding that the presence of highly aerenchymatous roots of Juncus maritimus in waterlogged soil increases the ability of Centaurium littorale and Plantago coronopus to endure prolonged submergence [25]. Meanwhile, Yang et al. [26] reported that competition between C. operculatus and Syzygium jambos under well-watered conditions can be transformed into facilitation under waterlogging conditions. In contrast to these results, rapid adventitious root growth can significantly enhance the competitiveness of Bidens pilosa under waterlogging conditions [27]. These contradictory results indicate that the mechanisms of plant–plant interactions under waterlogging conditions are incompletely understood.

In addition, physiological differences among neighboring plants are a prerequisite for complementary or facilitated resource use, which might act as a possible underlying mechanism affecting plant–plant interaction [28]. An extensive series of experiments have confirmed that N fixed by legumes can be transferred to nonlegumes, and this additional N source has positive effects on the growth of neighboring plants in mixed or intercropping systems [29]. These plants include Oryza sativa, Triticum aestivum, Lolium perenne, Cichorium intybus, Eucalyptus urophylla, and Eucalyptus grandis [30,31,32]. By contrast, a limited number of studies have found that legumes adversely affect survival and growth of Prunus serotina, Quercus rubra, Juglans nigra, Liriodendron tulipifera, and Fraxinus americana [33]. Unfortunately, the effects of waterlogging and legumes species on neighboring plants have not been comprehensively investigated.

C. operculatus and D. odorifera are tropical terrestrial trees belonging to the families Myrtaceae and Leguminosae, respectively, which are mainly distributed in south China and other tropical regions in the world [17,34]. They are widely used in clinical and basic laboratory studies focusing on the prevention and treatment of various disorders [17,35]. Our previous studies confirmed that they can be used in constructing riparian protective forests [17,36,37], but the effects of waterlogging stress on the neighboring relationships between C. operculatus and D. odorifera have still not been explored. The responses of C. operculatus to waterlogging frequently involve the production of adventitious roots with large amounts of internal oxygen, and the N-fixation capacity of legumes is extremely sensitive to changes in oxygen concentration in soil [32,38]. Therefore, we tested the following hypotheses: (i) under well-watered conditions, C. operculatus can directly benefit from the presence of D. odorifera; (ii) waterlogging stress can alter pre-existing species interactions and create a mutualistic relationship between two species. To test our hypotheses, we quantified changes in morphologic traits, biomass accumulation, and related physiological and biochemical parameters of C. operculatus and D. odorifera saplings. These results can provide a valuable reference for screening waterlogging-tolerant arbor species and species collocation during vegetation restoration and reconstruction activities in wetland systems.

2. Materials and Methods

2.1. Plant Materials and Experimental Designs

Two year old saplings of D. odorifera and C. operculatus were purchased from Jianfengling (18°39′ N, 108°44′ E) and Zengcheng (23°29′ N, 113°82′ E), respectively, in October 2020. To ensure uniform growth after re-tillering, we cut each sapling at 5 cm above the soil surface. The roots were washed carefully with tap water, and then replanted in plastic pots (49 cm in length, 14 cm in width, and 19 cm in height) containing 15 kg of sandy soil (sand: red soil = 2:1, v/v). Two saplings were planted 20 cm apart in each pot. The soil physicochemical properties, annual mean rainfall, temperature, and insulation time in the study area were described in our previous publication [17]. All pots with saplings were placed in a well-ventilated greenhouse at the experimental field of Hainan University. In late May 2021, saplings with the same crown size and equal height were selected for subsequent waterlogging experiments. The experimental layout was completely randomized and consisted of combined watering and planting patterns. The two watering treatments were well-watered and waterlogging, and the three planting patterns were D. odorifera + D. odorifera, C. operculatus + C. operculatus, and D. odorifera + C. operculatus. For each species, the well-watered and waterlogging treatments in the single planting pattern were marked as CK and W, respectively. Then, these treatments were marked as CK-M and W-M in the mixed planting patterns, respectively. For CK and CK-M treatments, we watered the saplings regularly with tap water to bring the soil water content close to the field capacity. For W and W-M treatments, the saplings were partially submerged in a large plastic box filled with tap water (flooded 10 cm above soil surface). A total of 15 pots (five replicates and three pots per replicate) were used for each planting pattern in each watering regime. Waterlogging experiments were started on 31 May 2021 and lasted until the plants were harvested on 10 August 2021.

2.2. Analyses of Morphological Traits and Tissue Biomass Accumulation

At the end of the experiment, all saplings were sampled and divided into leaves, stem, primary roots, and adventitious roots (only in C. operculatus) after they were carefully washed with tap water to remove sticking soil. Then, the adventitious root number (ARN), increment of stem height (SHI), leaf number (LNI), adventitious root fresh weight (ARFW), underground fresh weight (UFW), stem fresh weight (SFW), leaf fresh weight (LFW), and total plant fresh weight (TPFW) were measured or calculated for each plant. The total leaf area (TLA) in each plant was determined using an LI-3000 C Area Meter (Li-Cor Inc., Lincoln, NE, USA).

2.3. Determination of Gas Exchange, Chlorophyll Fluorescence, and Photosynthetic Pigment Content

From 8:30 a.m. to 11:30 a.m. on 9 and 10 August 2021, the net photosynthetic rate (A), stomatal conductance (g_s), and transpiration rate (E) were measured on the youngest fully expanded leaves of each sapling with an open gas exchange system (LI-6400XT, LI-COR Inc.): temperature, 30 °C; light intensity, 1400 μmol photons·m⁻²·s⁻¹; relative humidity, 60%; ambient CO₂, 350 ± 5 μmol·mol⁻¹. Chlorophyll fluorescence measurements were carried out on the same leaves used for gas exchange analyses with a modulated fluorometer (Junior-PAM; Walz, Effeltrich, Germany) according to the protocol described by Li et al. [17]. Photosynthetic pigment content was determined in the leaves previously used for the chlorophyll fluorescence measurements. Briefly, chlorophylls were extracted in 80% (v/v) chilled acetone. The absorbance of chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Caro) was recorded at 663, 646, and 470 nm, respectively. Chlorophyll concentration was calculated using the equations from Lichtenthaler [39], and the total chlorophyll (Tchl) was calculated as the sum of Chla and Chlb.

2.4. Determination of Soluble Protein, Free Proline, Peroxidase, and Superoxide Dismutase

The content of soluble protein in leaves was determined at an absorbance of 595 nm using the Bradford method [40], and soluble protein content was estimated from the standard curve of bovine serum albumin. The content of free proline in the leaves was measured spectrophotometrically (UV-1800PC, Shanghai Mapada Instruments Co., Ltd., Shanghai, China) at a wavelength of 520 nm with acid ninhydrin reagent, and Pro content was expressed as μg·g⁻¹ FW [41]. Peroxidase (POD) activity in the leaves was measured as the change in absorbance at 470 nm with guaiacol and H₂O₂ as substrates according to our previously described method [17]. POD activity was expressed as unit·min⁻¹·g⁻¹ FW. Superoxide dismutase (SOD) activity in the leaves was determined using nitroblue tetrazolium chloride, as reported previously by Li et al. [17]. SOD activity was expressed as unit·min⁻¹·g⁻¹ FW.

2.5. Determination of Superoxide Radical, H₂O₂, Malondialdehyde, Soluble Sugar, Starch, Primary Root Activity, and Midday Leaf Water Potential

Superoxide radical (O₂^·−), H₂O₂, malondialdehyde (MDA), soluble sugar, starch, and primary root activity were measured as described by our previous reports [17]. Midday leaf water potential (Ψ md) was measured in the leaf used for the gas exchange and chlorophyll fluorescence determination with a potentiometer (WP4C; Decagon Devices, Inc., Pullman, WA, USA) according to a previously described protocol [42].

2.6. Determination of Glutathione and Ascorbic Acid

The concentration of reduced glutathione (GSH) was measured in accordance with the method of Guri with some modifications [43]. Fresh leaf samples (1.0 g) were homogenized with 25 mL of 5 M EDTA-TCA in an ice bath and subjected to centrifugation at 12,000 rpm and 4 °C for 5 min. Approximately 2 mL of the supernatant was transferred to a test tube and titrated with 0.4 mL of 1 M NaOH to a pH range of 6.5–7.0. Then, 0.5 mL of 0.2 M potassium phosphate buffer (pH 7.0) and 0.1 mL of the reagent dithiobis-2-nitrogenzoic acid were added. After incubation at 25 °C for 5 min, distilled water was added to adjust the final volume to 5 mL. Absorbance was recorded at 412 nm; GSH was used as the calibration curve in calculating GSH concentration and expressed as μg·g⁻¹ FW. Ascorbic acid was measured as described by Kittipornkul [44]. Fresh leaf samples (0.1 g) were ground in a prechilled mortar and pestle with 4 mL of 6% trichloroacetic acid. After centrifuging at 12,000 rpm, 4 °C for 10 min, the precipitate was discarded, and 6% trichloroacetic acid was added to adjust the final volume to 4 mL. The supernatant was mixed with 2% dinitrophenyl hydrazine (2 mL), and then one drop of 10% thiourea solution (in 70% ethanol) was added. The mixture was heated in boiling water for 15 min and then rapidly cooled in an ice bath. Then, 5 mL of 80% (v/v) H₂SO₄ was added to the mixture. Absorbance was measured at 530 nm with a spectrophotometer.

2.7. Comprehensive Evaluation of Plant Growth among Different Treatments

Principal component analysis (SPSS 25.0) was carried out on all the indices of D. odorifera and C. operculatus for dimension reduction. If the accumulated variance represented over 85% of the total variance, the remaining components could be ignored. The membership function value of each selected component was calculated according to the method of Yan et al. [45]. The weight of each selected component was obtained using the following formula:

$$Wi=\frac{ci}{{{\displaystyle \sum }}_{i=1}^{n}ci}i=1, 2, 3,\dots , n,$$

where Wi represents the weight of the i-th principal component, and ci is the variance of i-th principal component obtained by PCA. The final comprehensive evaluation (CE) value was obtained using the method of Xiang et al. [46].

2.8. Statistical Analysis

SPSS 25.0 (SPSS, Chicago, IL, USA) was used in performing statistical analyses. Data were checked for normality and homogeneity of variances prior to the analysis and Ln-transformed when these assumptions were not satisfied. One-way ANOVA was used in determining differences between treatments, and Duncan’s multiple range test was used in detecting possible differences among means. Two-factor ANOVA with LSD post hoc tests was used in determining the effects of waterlogging, planting, and their combinations. Differences were considered significant at the p < 0.05 level.

3. Results

3.1. Comparative Analysis of Growth Traits in Both Species under CK, CK-M, W, and W-M Conditions

As shown in Figure 1, D. odorifera had better growth performance (SHI, LNI, and TLA) than C. operculatus under CK conditions. Compared with CK, CK-M showed a significant decrease in SHI and LNI (71.43% and 49.25%) in D. odorifera, as well as significant increases in SHI and TLA (61.86% and 33.87%) in C. operculatus. W treatment significantly decreased SHI, LNI, and TLA in D. odorifera, and significantly increased the SHI (69.59%) in C. operculatus. Compared with CK-M treatment, W-M significantly decreased the SHI, LNI, and TLA in D. odorifera and significantly increased TLA in C. operculatus. Compared with W treatment, W-M significantly decreased SHI and ARN and significantly increased TLA and ARFW in C. operculatus. In addition, W and W-M treatments induced AR formation in C. operculatus but not in D. odorifera.

3.2. Comparative Analysis of Biomass Accumulation in Both Species under CK, CK-M, W, and W-M Conditions

After 70 days of treatment, D. odorifera always showed a significantly higher SFW, LFW, and TPFW and significantly lower levels of UFW than C. operculatus in the CK treatments (Figure 2). Compared with CK, CK-M treatment significantly decreased SFW and TPFW in D. odorifera and significantly increased SFW (40%) in C. operculatus. W treatment significantly decreased SFW, LFW, UFW, and TPFW in D. odorifera (36.30%, 73.36%, 58.58%, and 59.44%, respectively) and significantly increased UFW and TPFW (35.00% and 37.35%, respectively) in C. operculatus. Compared with CK-M, W-M treatment significantly decreased SFW, LFW, UFW, and TPFW (86.83%, 61.53%, 63.16%, and 72.00%, respectively) in D. odorifera saplings and significantly increased UFW and TPFW in C. operculatus. In addition, compared with W, W-M treatment significantly decreased SFW and TPFW in D. odorifera and significantly increased LFW in C. operculatus.

3.3. Comparative Analyses on Chlorophyll Content in Both Species under CK, CK-M, W, and W-M Conditions

As shown in Figure 3, compared with CK treatment, CK-M treatment significantly increased Chla, Chlb, TChl, and Caro content in C. operculatus and decreased Caro content in D. odorifera. Obviously, mixed planting had more positive effects on Chla, Chlb, and TChl content in C. operculatus than in D. odorifera. Compared with CK treatment, W treatment had significant negative effects on Chla, Chlb, and TChl content in both species. W treatment significantly decreased Caro content in D. odorifera but significantly increased it in C. operculatus. Variations in the trends of pigment content between W-M and CK-M treatments were the same as those between W and CK treatments. Compared with W treatment, W-M treatment had little effects on all parameters in both species, except Caro content in C. operculatus.

3.4. Comparative Analysis on Photosynthetic Characteristics in Both Species under CK, CK-M, W, and W-M Conditions

At the end of the experiment, the highest photosynthetic values of D. odorifera and C. operculatus were detected in CK and CK-M treatments, respectively (

Table 1. Comparative analyses of the parameters of gas exchange and chlorophyll fluorescence of C. operculatus and D. odorifera under all treatments.

	Treatment	A (μmol·m−2·s−1)	gs (mol·m−2·s−1)	E (mmol·m−2·s−1)	Fv/Fm
D. odorifera	CK	13.94 ± 0.73 a	0.23 ± 0.04 a	5.70 ± 0.89 a	0.85 ± 0.01 a
	CK-M	10.82 ± 0.83 b	0.19 ± 0.05 a	4.56 ± 1.06 a	0.83 ± 0.00 a
	W	2.81 ± 0.24 c	0.07 ± 0.01 b	2.23 ± 0.12 b	0.64 ± 0.02 c
	W-M	4.27 ± 0.99 c	0.09 ± 0.00 b	2.35 ± 0.02 b	0.74 ± 0.02 b
FW		0.000	0.001	0.002	0.000
FP		0.293	0.693	0.478	0.012
FW*P		0.010	0.390	0.383	0.001
C. operculatus	CK	12.71 ± 1.21 AB	0.13 ± 0.04 A	4.19 ± 1.25 B	0.83 ± 0.01 AB
	CK-M	18.66 ± 5.47 A	0.21 ± 0.06 A	8.45 ± 1.80 A	0.85 ± 0.00 A
	W	5.82 ± 0.68 B	0.10 ± 0.00 A	3.22 ± 0.14 B	0.83 ± 0.01 AB
	W-M	11.31 ± 0.99 A	0.13 ± 0.01 A	4.21 ± 0.38 B	0.82 ± 0.00 B
FW		0.029	0.157	0.038	0.073
FP		0.069	0.174	0.036	0.448
FW*P		0.938	0.566	0.168	0.073

A, net photosynthesis rate; g_s, stomatal conductance; E, transpiration; Fv/Fm, maximum photochemical quantum yield of PSII. Abbreviations, explanations of treatments, data descriptions, and statistics are the same as those shown in Figure 1.

). Compared with CK treatment, CK-M treatment significantly decreased A in D. odorifera and increased A, g_s, E, and Fv/Fm in C. operculatus, while E was significantly increased. W treatment significantly reduced A (79.84%), g_s (69.57%), Fv/Fm (23.81%), and E (60.88%) in D. odorifera but had insignificant effects on these parameters in C. operculatus. Compared with CK-M treatment, W-M treatment significantly reduced the A (69.37%), g_s (60.87%) Fv/Fm (12.05%), and E (48.46%) in D. odorifera and significantly deceased Fv/Fm and E in C. operculatus. Compared with W treatment, W-M treatment only significantly increased the Fv/Fm level in D. odorifera.

3.5. Comparative Analysis on Antioxidant Enzymes and Osmoregulatory Substances in Both Species under CK, CK-M, W, and W-M Conditions

Significant differences in soluble protein content and SOD activity were found between D. odorifera and C. operculatus in all treatments, and D. odorifera always showed a significantly higher soluble protein content but significantly lower SOD activity than C. operculatus within the same treatment (

Table 2. Comparative analyses of the contents of soluble protein, free proline, SOD, and POD of C. operculatus and D. odorifera under all treatments.

	Treatment	SP (mg·g−1·FW)	Pro (μg·g−1·FW)	POD (U·g−1·FW·h−1)	SOD (U·g−1·FW·h−1)
D. odorifera	CK	27.07 ± 0.64 b	13.42 ± 0.49 b	230.59 ± 26.14 c	82.70 ± 29.75 b
	CK-M	26.34 ± 1.25 b	12.24 ± 1.99 b	367.75 ± 13.19 b	120.08 ± 21.96 b
	W	34.11 ± 0.95 a	26.60 ± 0.79 a	663.44 ± 71.66 a	422.23 ± 11.35 a
	W-M	31.44 ± 1.28 a	25.44 ± 1.27 a	657.79 ± 22.90 a	395.98 ± 33.55 a
FW		0.000	0.000	0.000	0.000
FP		0.136	0.374	0.129	0.832
FW*P		0.378	0.996	0.102	0.238
C.operculatus	CK	14.85 ± 0.24 B	17.46 ± 0.15 B	161.25 ± 19.96 C	653.74 ± 50.17 C
	CK-M	11.73 ± 0.73 C	18.56 ± 0.36 B	255.62 ± 11.49 AB	705.80 ± 64.93 C
	W	27.24 ± 0.96 A	26.94 ± 1.22 A	219.31 ± 9.94 B	1328.13 ± 19.30 A
	W-M	26.25 ± 1.49 A	25.42 ± 1.04 A	280.37 ± 18.73 A	998.70 ± 17.12 B
FW		0.000	0.000	0.021	0.000
FP		0.055	0.802	0.000	0.007
FW*P		0.290	0.138	0.308	0.001

SP, soluble protein; Pro, free proline; POD, peroxidase; SOD, superoxide dismutase. Abbreviations, explanations of treatments, data descriptions, and statistics are the same as those shown in Figure 1.

). Compared with CK, CK-M treatment significantly increased POD activity in both species and significantly decreased soluble protein content in C. operculatus. W treatment significantly increased the soluble protein and free proline content and POD and SOD activities in both species. Compared with CK-M, W-M treatment significantly increased soluble protein and free proline content, as well as SOD activity, in both species and POD activity in D. odorifera. Moreover, W-M treatment significantly increased POD activity and decreased SOD activity in C. operculatus but had insignificant effects on the same parameters in D. odorifera compared with W treatment.

3.6. Comparative Analysis on GSH and ASA Content in Both Species under CK, CK-M, W, and W-M Conditions

C. operculatus had higher ASA content than D. odorifera in all treatments (Figure 4). Compared with CK treatment, CK-M treatment only caused a significant decrease in ASA content in C. operculatus, whereas it had insignificant effects on GSH content in both species and ASA content in D. odorifera. W treatment significantly increased ASA and GSH content in both species. Compared with CK-M treatment, W-M treatment significantly increased the content of GSH and ASA in C. operculatus saplings and significantly increased GSH content in D. odorifera saplings. Furthermore, W-M treatment significantly decreased ASA content in both species but had insignificant effects on GSH content in both species compared with the W treatment.

3.7. Comparative Analysis on Nonstructural Carbohydrate Content and Activity in Primary Roots of Both Species under CK, CK-M, W, and W-M Conditions

In general, significantly higher level in root activity was found in D. odorifera than in C. operculatus within the same treatment, and a significantly higher level in soluble sugar content and lower level in starch content were found in D. odorifera than in C. operculatus under waterlogging condition (Figure 5). Compared with CK treatment, CK-M treatment significantly increased soluble sugar content and root activity in C. operculatus and significantly reduced root activity in D. odorifera. W treatment significantly increased soluble sugar and starch content but significantly reduced root activity in both species. Compared with CK-M treatment, W-M significantly increased the soluble sugar and starch content in D. odorifera and starch content in C. operculatus and significantly reduced root activity in C. operculatus. Differences in soluble sugar content, starch content, and root activity between W and W-M treatments were nonsignificant within each species.

3.8. Comparative Analysis on Reactive Oxygen Species, Cell Membrane Permeability, and Midday Leaf Water Potential in Both Species under CK, CK-M, W, and W-M Conditions

Significant interspecific differences in H₂O₂ and MDA levels were found in all treatments, and D. odorifera exhibited a higher level of MDA content but lower level of H₂O₂ content than C. operculatus (

Table 3. Comparative analyses of the levels of O₂^·−, H₂O₂, MDA, and Ψ_md of C. operculatus and D. odorifera under all treatments.

Species	Treatment	O2·− (μg·g−1·FW)	H2O2 (mol·g−1)	MDA (μmol·g−1·FW)	Ψmd (MPa)
D. odorifera	CK	210.73 ± 16.34 b	6.47 ± 0.44 b	17.34 ± 0.55 b	−3.76 ± 0.11 a
	CK−M	178.35 ± 10.04 b	5.06 ± 0.90 b	20.95 ± 1.19 b	−3.70 ± 0.14 a
	W	508.25 ± 14.89 a	12.24 ± 0.44 a	33.95 ± 1.32 a	−6.85 ± 0.73 b
	W−M	223.32 ± 33.50 b	6.63 ± 1.09 b	36.08 ± 2.39 a	−6.34 ± 0.56 b
FW		0.000	0.000	0.000	0.000
FP		0.000	0.001	0.083	0.562
FW*P		0.000	0.018	0.633	0.644
C.operculatus	CK	202.41 ± 47.64 AB	100.58 ± 5.14 BC	9.65 ± 0.18 C	−4.20 ± 0.42 A
	CK−M	143.82 ± 37.96 B	96.72 ± 4.90 C	8.02 ± 0.66 D	−4.11 ± 0.40 A
	W	271.93 ± 33.21 A	163.83 ± 3.97 A	13.79 ± 0.38 A	−4.93 ± 0.17 A
	W−M	184.77 ± 13.79 AB	113.47 ± 3.54 B	11.61 ± 0.41 B	−4.47 ± 0.15 A
FW		0.144	0.000	0.000	0.103
FP		0.062	0.000	0.001	0.399
FW*P		0.693	0.000	0.551	0.564

O₂^·−, superoxide radicals; H₂O₂, hydrogen peroxide; MDA, malondialdehyde; Ψ_md, leaf midday water potential. Abbreviations, explanations of treatments, data description, and statistics are the same as those shown in Figure 1.

). Compared with CK treatment, CK-M treatment significantly decreased MDA content in C. operculatus but had little effects on other parameters in both species. W treatment significantly increased O₂^·− (58.54%), MDA, and H₂O₂ content in D. odorifera, and only significantly increased H₂O₂ and MDA content in C. operculatus. Compared with CK-M treatment, W-M treatment significantly increased MDA content in both species and H₂O₂ content in C. operculatus. Compared with W treatment, W-M significantly reduced O₂^·− and H₂O₂ content in the D. odorifera and significantly reduced H₂O₂ and MDA content in C. operculatus. In addition, waterlogging stress caused a significant reduction in leaf water potential in D. odorifera but did not in C. operculatus.

3.9. Comparative Comprehensive Evaluation among Different Treatments

Comparative analyses on the value of the principal component [C(µ)], membership function value [M(µ)], and comprehensive evaluation value (CE) between C. operculatus and D. odorifera in the treatments are shown in

Table 4. Comparative analyses of the value of the principal component [C(µ)], membership function value [M(µ)], and comprehensive evaluation value (CE) of C. operculatus and D. odorifera under all treatments.

Species	Treatment	C (1)	C (2)	C (3)	M (1)	M (2)	M (3)	CE
D. odorifera	CK	0.973	−1.113	1.228	0.944	0.000	1.000	0.630
	CK-M	0.506	−1.073	0.489	0.769	0.015	0.741	0.509
	W	−1.390	−0.517	−0.513	0.057	0.221	0.391	0.153
	W-M	−1.543	−0.537	−0.091	0.000	0.213	0.538	0.137
C. operculatus	CK	0.408	0.408	−1.048	0.732	0.563	0.203	0.611
	CK-M	1.122	0.078	−1.629	1.000	0.441	0.000	0.690
	W	−0.204	1.586	0.720	0.502	1.000	0.822	0.710
	W-M	0.128	1.169	0.844	0.627	0.845	0.866	0.730

Abbreviations and explanations of treatments are the same as those shown in Figure 1.

. Fresh weight, and morphological and physiological traits were reduced to three principal components that explained 92.84% of the variance. D. odorifera had higher CE values than C. operculatus in CK treatments. The CE value of D. odorifera sequentially reduced in the CK, CK-M, W, and W-M treatments, but completely opposite trends were found in C. operculatus. C. operculatus from W-M treatment possessed the highest CE value, and C. operculatus always had higher CE values than D. odorifera under waterlogging conditions.

4. Discussions

4.1. Growth of C. operculatus Was Clearly Promoted by the Presence of D. odorifera under Well-Watered Condition

In this study, we found that well-watered mixed-planting treatment had a negative effect on the growth of D. odorifera and a positive effect on C. operculatus (Figure 1a and Figure 2a,d; Table 4). These results suggest that neighboring D. odorifera had a net facilitating effect on C. operculatus mainly because D. odorifera can obtain nitrogen through symbiotic N₂ fixation. The nonlegumes of C. operculatus can benefit from the increase in soil nitrogen availability in mixed planting [47]. Evans stated that nitrogen level is critical for chlorophyll biosynthesis, and any plant species with high chlorophyll content would have an advantage in light absorption [48,49]. The increase in chlorophyll content can increase the efficiency of C. operculatus in converting light energy into chemical energy, thereby improving the photosynthetic rate and ultimately increasing biomass (Figure 3a–c). This observation is strengthened by the higher total leaf area observed in C. operculatus (Figure 1c). In addition, some researchers noted that carotenoids play a vital role in preventing the chlorophyll-photosensitized formation of ¹O₂ [50]. High carotenoids can enhance the protection of chloroplast membrane integrity in C. operculatus (Figure 3d and Table 3). Munns et al. reported that a decline in osmotic adjustment substances increases the osmotic potential of plant root cells and decreases the water absorption capacity of the root [51]. Consequently, well-watered mixed-planting treatment significantly increased root activity and soluble sugar content of C. operculatus, indicating that primary roots had stronger water and nutrient absorption capacities. Overall, the growth of C. operculatus was clearly promoted by the presence of D. odorifera under well-watered conditions.

4.2. C. operculatus Was More Tolerant to Waterlogging Than D. odorifera

Long-term waterlogging did not result in the death of any plant, suggesting that two species are waterlogging-tolerant terrestrial woody plants. However, C. operculatus exhibited better waterlogging tolerance (Figure 2d and Table 4). Waterlogging tolerance in plants usually depends on a combination of metabolic and morphological responses [52]. The ability to form well-developed adventitious roots and leaves is important to O₂ diffusion, water and nutrient uptake, and light capture. Therefore, C. operculatus with a large number of adventitious roots and high leaf area and number would be at an advantage (Figure 1d). Meanwhile, many plants respond to waterlogging by closing their stomata and downregulating the photosynthetic machinery, but both processes lead to the formation of reactive oxygen species (ROS) within the leaf because of light reactions [53]. The Fv/Fm value is considered a good indicator for damaging effects on PSII and can provide insights into the ability of plants to tolerate environmental stress and the extent to which stress can damage the light reaction center [17]. Under single-planting waterlogging conditions, the decrease in Fv/Fm was greater in D. odorifera than in C. operculatus, indicating that C. operculatus had a lower degree of damage to the light reaction center than D. odorifera. In general, damage to PSII under waterlogging stress is mainly attributed to the accumulation of ROS and damage to membrane lipids [54]. In the present study, the greater increases in O₂^·−, H₂O₂, MDA, SOD, POD, GSH, soluble protein, and free proline levels in D. odoriferax demonstrated that oxidative stress was more serious in D. odorifera (Figure 4a, Table 2 and Table 3). This result confirms the results obtained by Bhusal et al., who reported that proline, antioxidants, and secondary metabolites significantly increased in waterlogging-sensitive Pinus densiflora and Pinus thunbergii [55].

4.3. Competition to D. odorifera and Facilitation to C. operculatus Occurred under Mixed-Planting Waterlogging Condition

Previous studies emphasized the necessity for assessing intra- and interspecific relationships when studying the influences of abiotic factors on plant–plant interactions [14]. In this study, compared to well-watered mixed-planting treatment, we found that C. operculatus had an obvious inhibitory effect on the growth of D. odorifera, whereas D. odorifera significantly promoted the growth of C. operculatus under mixed-planting waterlogging conditions (Figure 2 and Table 4). C. operculatus benefitted by limiting its neighbors under waterlogging, probably through its ability to develop a large number of adventitious roots with aerenchyma. This ability enables the plant to compete for limited space, O₂, and nutrients from common-pool resources (Figure 1d). However, adventitious root formation was not observed in D. odorifera. In fact, this speculation was indirectly confirmed by Yue et al., who found that rapid adventitious root growth can significantly improve the competitiveness of B. pilosa under waterlogging [27]. The compensatory growth in total leaf area can be responsible for change in the growth patterns of C. operculatus (Figure 1c). The greater total leaf area of C. operculatus in mixed-planting waterlogging conditions can provide an adequate photosynthetic area for rapid assimilation and enable the plant to meet growth requirements, which are higher than those in single-planting waterlogging conditions [56]. Additionally, the phenomenon may be attributed to the lower increases in H₂O₂, MDA, SOD, POD, free proline, and GSH levels of C. operculatus (Figure 4a and Table 1, Table 2 and Table 3). C. operculatus has a better self-protective ability against ROS-induced cell toxicity [57]. Therefore, these alterations in morphological and physiological characteristics can collectively explain the changes in growth patterns in both species.

5. Conclusions

In the present study, we found that C. operculatus and D. odorifera are waterlogging-tolerant species, and that C. operculatus has higher tolerance to waterlogging than D. odorifera. C. operculatus was the beneficiary, whereas D. odorifera was the victim under well-watered mixed planting, and this effect could be strengthened by waterlogging. Overall, this study provided valuable information for screening waterlogging-tolerant arbor species and species collocation during vegetation restoration and reconstruction activities in wetland systems. However, the experiment was conducted in a greenhouse environment with inevitable limitations, and field work is being carried out to further verify our results.