Role of Stand Density in Shaping Interactions and Growth Strategies of Dioecious Tree Species: A Case Study of Fraxinus mandshurica

Abstract

Stand density is a primary limiting factor affecting the accumulation of timber volume, growth, and development of trees in plantations. However, the impact of stand density on the spatial structure and developmental strategies of male and female plants in dioecious tree species remains unclear. In this study, we focused on female, male, and unknown-sex plants of Fraxinus mandshurica across four initial densities (1 m × 1 m, 1.5 m × 1.5 m, 2 m × 2 m, 3 m × 1.5 m). From 2018 to 2022, continuous observations were conducted to determine sex and growth traits (tree height, diameter at breast height, and crown width) with measurements taken annually during the peak growing season. In 2022, in the same season, we measured the morphology and nutrient contents of vegetative organs (shoots, leaves, and absorptive roots) in plants of different genders and assessed the soil properties of their rhizosphere soil. The competition intensity among female plants at high density (D₄) increased significantly by 46.32% compared to low density. The gender mingling between female and male plants remained relatively stable across all densities and was greater than 0.7, and the plants occupied a sub-dominant position within their spatial structure. As density increases, the annual growth in height and crown width of female, male, and unknown-sex plants significantly decreases (p ≤ 0.05), while the annual timber volume growth of males and unknown-sex plants also experiences a significant reduction (p ≤ 0.05). Density was a primary factor affecting the ratio of the leaf area, branch thickness, diameter of the absorbing roots, and root tissue density in female and male plants. It also significantly influenced the changes in nitrogen (negatively) and phosphorus (positively) levels within the vegetative organs (p ≤ 0.05). Collectively, these changes were related to the moisture content, ammonium nitrogen, and total phosphorus levels in the rhizosphere soil. These findings emphasize the important of density and spatial structure in shaping the interactions between male and female plants, with the density influencing their growth and reproductive strategies. Research findings provide important insights into the cultivation strategies for dioecious tree species in plantations.

1. Introduction

The cultivation of plantation forests is one of the key methods for expanding global forest resources and ensuring the supply of timber products [1]. As of 2019, the total area of plantation forests in China reached 79.54 million hectares. By improving forest production management and selecting fast-growing and high-yield tree species, the rapid development of China’s forestry industry has been effectively promoted [2]. In timber forests, reasonable density and species selection play a crucial role in enhancing the overall quality of the stand [3].

Stand density is one of the important factors affecting the growth environment of individual trees in plantation forests [4]. It alters the light, temperature, humidity, and soil nutrients within the stand by adjusting its horizontal structure [5,6]. A higher stand density leads to intensified competition for resources among individuals, which may result in reduced growth rates and, subsequently, lower stand productivity [7]. Studies have shown that the competition for resources induced by density affects not only the productivity of male and female plants but also their numerical balance [8,9]. By considering the impact of stand density on the growth and development of dioecious tree species, forest managers may be able to enhance productivity while ensuring sustainable forest management practices [10].

The morphological differences between male and female dioecious plants are crucial for shaping their respective reproductive functions and growth patterns [11,12]. Research has indicated that male plants typically exhibit higher branch yields, thicker trunks, and a greater ability to produce lateral buds [13]. In contrast, female plants possess morphological characteristics that support fruiting, such as thicker nutrient branches, larger specific leaf weights, and a greater photosynthetic capacity [14]. Although female plants invest more nutrients into their fruits and seeds [15], they also exhibit compensatory mechanisms in aspects, such as photosynthetic rate and nutrient absorption, which can offset their reproductive costs [16]. Moreover, there are gender-specific differences in nutrient absorption and metabolism between male and female plants in dioecious species. For instance, Li discovered that female Populus deltoides plants exhibit selectivity for different forms of inorganic nitrogen—ammonium nitrogen and nitrate nitrogen—whereas male plants do not show any preference [17]. Chen [18] observed that in high-nitrogen environments, female Populus cathayana have a higher nitrogen utilization efficiency than males. However, it remains unclear how the spatial heterogeneity of growth, resulting from changes in stand density, impacts the dimorphic traits of female and male plants, as well as their strategies for nutrient uptake and storage.

The spatial structure of trees is defined as the basic unit of individual growth, consisting of the selected target tree and its four neighboring trees within the stand [19]. The nearest neighbor method can comprehensively evaluate the distribution pattern of tree spatial units through multiple indicators, such as the diversity index, competition index, and mixing degree [19,20]. This method helps to optimize the spatial layout of stands [21], promote the growth of target trees [22], and increase stand productivity [23]. The appropriate intensity of forest competition (growth space) significantly influences tree growth; however, upon reaching a certain threshold, an inhibitory effect among trees becomes evident [24]. The spatial unit composed of male and female trees and their surrounding neighbors plays a crucial role in development, wood formation, and seed production [25]. Studies have found that the reproductive process of mature dioecious plants affects the developmental status of neighboring trees [26,27]. Additionally, density and self-thinning cause changes in the spatial structure of forest trees and affect nutrient use efficiency in the soil [7]. These findings highlight the importance of considering fluctuations in spatial structure and their impact on forest dynamics when managing plantations. By considering the arrangement of trees within a stand, forest managers can make informed tending decisions.

In summary, existing studies have revealed widespread differences between female and male individuals in dioecious plants across aspects such as spatial structure, growth, leaves, shoots, root traits, stoichiometric characteristics of vegetative organs, and soil physical–chemical properties. Moreover, sexual dimorphism is influenced by environmental factors such as heterogeneity in spatial structure (size and composition). Under similar habitat conditions, the growth strategies of dioecious trees are more susceptible to spatial structural factors in addition to genetic factors, a phenomenon that becomes more pronounced after trees enter reproductive growth. However, the relationships among growth space size, structural composition heterogeneity, and density between female and male plants have not been systematically analyzed. Focusing on these questions, this study focuses on a plantation forest of F. mandshurica established by Professor Wang Qingcheng in 1998 in the eastern mountainous region of Heilongjiang Province, featuring different initial densities (spacing: 1 m × 1 m, 1.5 m × 1.5 m, 2 m × 2 m, 3 m × 1.5 m). We analyzed the spatial structure and growth conditions of F. mandshurica female and male trees at four different densities and explored the nutrient absorption strategies of individuals of different sexes through the physicochemical properties of rhizosphere soil. We aimed to answer the following questions: (1) Does density affect the spatial structure composition and growth capacity of female and male trees? and (2) Does density affect the morphology of the nutrient organs and the nutrient content of female and male trees, as well as the properties of the rhizosphere soil?

2. Materials and Methods

2.1. Study Area and Sites

The research site was located at the Maoershan Experimental Forest Farm of the Northeast Forestry University in Heilongjiang Province, China (127°29′–127°33′ E, 45°19′–45°24′ N). This location falls within the Zhangguangcai Ridge branch of Changbai Mountain and has an average altitude of approximately 300 m, characterized by low hills and slopes of less than 15 degrees, with dark brown soil. The regional climate exhibits a temperate continental monsoon climate pattern, featuring dry springs and autumns, brief summer rains, and cold winters. Annual precipitation ranges from 700 to 750 mm, while the average annual temperature hovers between 2.7 and 2.9 °C. The vegetation in this region is part of the Changbai Mountain flora region, with the predominant forest community consisting of a mixed forest of Korean pine and broad-leaved trees [28].

The experimental plot for this study was established in 1998, where 2-year-old F. mandshurica seedlings were planted after clear-cutting. Strip afforestation was conducted at four different densities (spacing: 1 m × 1 m, 1.5 m × 1.5 m, 2 m × 2 m, 3 m × 1.5 m). The slope faces east, and the forests are of the same age, with similar understory vegetation and identical climate conditions, making it a suitable growing area for F. mandshurica. Within each density, three standard plots measuring 20 m × 30 m were set up as replicates, with intervals exceeding 15 m (Figure 1).

2.2. Spatial Structure Measurement

In early July 2022, during the vigorous growth period, well-developed (vigorous growth and free of pests and diseases) individuals (6 males, 6 females, and 6 individuals of unknown gender, totaling 72 trees at each density) were selected for investigation and sampling at the standard site. The selected target trees were representative of the growth and development status of different gender individuals within each density, and the sample size met the requirements for subsequent statistical analysis. Each spatial unit consisted of one target tree and four neighboring trees. This study utilized four indicators—Gender mingling, the Opening degree index, the Hegyi competition index, and Neighborhood comparison [19]—to describe the spatial structure of the reproductive and unknown gender plants of F. mandshurica based on actual conditions. The improved calculation formulas and their explanations are as follows.

The degree of gender mingling between the target tree and the neighboring trees in the spatial structural unit is represented by the following formula [19]:

$$M_i={\displaystyle \frac{1}{n}}\sum _{j=1}^n{V}_{ij}$$

In the formula, the n represents the number of neighboring trees (in this study, it was set to 4). If the j-th neighboring tree of the i-th target tree has the same gender, the value of V_ij is 0; otherwise, the value of V_ij is 1. The larger the value of M_i, the greater the degree of gender mixing in the structural unit composed of the n neighboring trees.

This parameter reflects the light transmission of trees inside the forest. It quantifies the ratio of the horizontal distance between the trees inside the forest and their height to the adjacent trees. The formula is as follows [19]:

$$K_i={\displaystyle \frac{1}{4}}\sum _{j=1}^4{\displaystyle \frac{D_{ij}}{H_j}}$$

In the equation, the D_ij represents the horizontal distance between tree i and the adjacent tree j; the H_j represents the height of the adjacent tree j. For the K_i ∈ (0, +∞), the larger the value, the better the light transmission of tree i.

This reflects the competitive intensity between the target tree and the neighboring tree, and the formula is as follows [19]:

$$C_H={\displaystyle \frac{1}{n}}\sum _{j=1}^n{\displaystyle \frac{D_j}{D_i}}{\displaystyle \frac{1}{d_{ij}}}$$

In the formula, the D_j represents the diameter of the competition tree j, D_i represents the diameter of the object tree i. d_ij represents the distance between the object tree i and the competition tree j. A higher C_H value indicates a more intense competition between trees.

This is defined as the proportion of neighboring trees with a breast diameter greater than the target tree among their neighbors. The formula is as follows [19]:

$$U_i={\displaystyle \frac{1}{n}}\sum _{i=1}^n{K}_{ij}$$

In the formula, when the diameter of the adjacent tree j is greater than the diameter of the target tree i, K_ij equals 1; otherwise, K_ij equals 0. The U_i = 0 indicates that the object tree is in an absolute dominant position in the spatial structural unit; the 0 < U_i < 0.25 suggests a subdominant position; the 0.25 < U_i < 0.5 indicates a moderate position; the 0.5 < U_i < 0.75 represents a disadvantageous position; and the 0.75 < U_i < 1 signifies an absolute disadvantageous position.

2.3. Growth Traits Measurement

From 2018 to 2022, selected plants at different densities were continuously observed, with a total of 288. The sample size adequately represents the growth status of F. mandshurica at varying densities. Crown width data were recorded during the peak of the growing season (mid-July), while tree height and diameter at breast height were measured after the end of the growing season (end of October each year). The specific measurement methods and descriptions of each indicator are as follows.

The tree height was measured using a height and distance instrument (Vertex IX, Stockholm, Sweden). A DBH ruler was used to measure the trunk diameter 1.3 m above the ground. The east-west and north-south dimensions of the tree crown were quantified with a tape measure, and the average was calculated to determine the crown width. The tree volume of timber was calculated using a two-variable equation with the specified formula [29]:

Y = 0.000048 × D^2.01077 × H^0.870343

The D is the tree diameter at breast height, and the H is the tree height.

2.4. Phenotype and Nutrients of Nutritive Organ

The plant organs selected for measuring morphological and physiological indicators included leaves, annual branches, and absorptive roots. The morphological measurements encompassed specific leaf area, leaf shape index, annual branch length and thickness, root mean diameter, specific root length, specific root surface area, and root tissue density. The chemical indicators measured include total carbon, nitrogen, phosphorus, and potassium (measurement methods are detailed below).

For each selected reproductive and unknown gender plant, branches and leaves were harvested from the southern-facing middle of the crown using high branch shears. The leaves and shoots were collected, placed in sealed bags, kept fresh in a portable refrigerator, and transported back to the laboratory. The length and width of three leaflets from the top of each compound leaf were measured using vernier calipers (30 leaflets per plant) to calculate the leaf shape index [30]. Fresh leaves were punched with a 10 mm diameter hole punch, taking care to avoid the veins, followed by high-temperature fixation at 105 °C for 30 sec and then oven drying at 65 °C to determine biomass, subsequently allowing for the calculation of the specific leaf area. The length and thickness of the shoots were evaluated with vernier calipers, selecting 10 one-year-old branches from each tree. Absorptive roots (levels 1–3) were excavated in the 0–20 cm soil layer, half a meter away from the trunk and in the direction of root extension using a shovel [31]. Multiple complete root segments were placed in numbered ziplock bags for analysis (6 bags for each gender in each density, totaling 144 bags). Root system scanning was performed with an Epson digital scanner (Expression 10000XL 1.0) to capture images. Subsequently, WinRhizo 2004b software (Regent Instruments Inc., Québec City, QC, Canada) was utilized, along with the weighted average method, to compute the root length, surface area, average diameter, and volume of the absorptive roots. The dry matter content was determined by drying at 65 °C until a constant weight was reached. Specific root length, root tissue density, and specific surface area were then computed as outlined by Ren [32].

Plant samples, comprising leaves, branches, and absorptive roots, were collected and analyzed for nutrients. Female and male samples from each standard plot were grouped into a single replicate, with three experimental replicates for each density. Total carbon and nitrogen in the vegetative organs were assessed using a carbon and nitrogen analyzer (Vario Macro, Nidderau, Germany), total potassium was measured with a flame spectrophotometer, and total phosphorus was analyzed using a continuous flow injector (BRAN+LUEBBE-AA3, Norderstedt, Germany).

2.5. Physicochemical Properties of Rhizosphere Soil

The shake-off method was used to sample rhizosphere soil (0–20 cm soil layer) from reproductive and unknown gender individuals at different densities [33]. The soil samples obtained were carefully sealed in ziplock bags and transported in an icebox to the laboratory. Upon arrival, the soil was segregated by gender and field location, with plant residues and stones removed before evenly dividing the soil into two sub-samples. One portion was naturally air-dried, while the other was frozen and stored at −20 °C for future soil analysis. Various physical and chemical properties of the soil were analyzed, including pH, moisture content, ammonium nitrogen, nitrate nitrogen, total carbon, total nitrogen, total phosphorus, and total potassium content. The specific measurement methods for each indicator were as follows.

Soil moisture content and pH were determined by weighing equal amounts of fresh soil samples (10 g, accurate to 0.001 g) and drying them at 65 °C until a constant weight was achieved. Subsequently, the moisture content of the samples was calculated while maintaining a soil-to-water ratio of 1:2.5. The soil pH was measured using a pH meter (PHS-3C, Hangzhou, China). NH₄-N and NO₃-N in the soil were extracted with a 2 mol·L⁻¹ potassium chloride solution and analyzed using a flow analyzer (BRAN+LUEBBE-AA3, Germany). Total carbon, nitrogen, phosphorus, and potassium were measured using the same instruments as those used for the plants.

2.6. Statistical Analysis

The data were organized using Microsoft Excel 2021 (Redmond, WA, USA). Before analyzing the data, SPSS 26.0 (SPSS Corp., Armonk, NY, USA) was used to check the data distribution characteristics to determine whether they conformed to a normal distribution and homogeneity of variances. One-way analysis of variance was performed to evaluate the impact of density on the morphology of female and male trees, vegetative organs, and soil nutrients. Two-way analysis of variance was applied to investigate the effects of density and gender on the aforementioned indicators. LSD’s multiple test was employed for multiple comparisons to determine significance. Pearson’s correlation coefficient was calculated to evaluate the relationship between the indicators of female and male trees. Furthermore, a Mantel test was performed to assess the influence of spatial structure on growth indicators. Data analysis and visualization were carried out using Origin 2021 and the ggplot2 package in R (version 4.2.2).

3. Results

3.1. Spatial Structure

The competition intensity among female plants at high density (D₄) increased significantly by 46.32% compared to low density (D₁, p ≤ 0.05). Additionally, the competition indices of unknown-sex plants within densities D₁ to D₃ were significantly greater than those of reproductive plants (p ≤ 0.05, Figure 2A). There were no significant differences in the opening degree indices among different sexes either between or within densities (Figure 2B). The gender mingling between female and male plants remained relatively stable across all densities and was greater than 0.7, indicating the presence of same-sex repulsion. The gender mingling of unknown-sex plants at density D₁ was significantly greater than that of the other densities (p ≤ 0.05). Except for density D₁, the gender mingling of reproductive plants in the other densities was significantly greater than that of unknown-sex plants (p ≤ 0.05, Figure 2C). Female and male plants were both in a sub-dominant position or higher within their respective spatial structure units, while unknown-sex plants occupied a sub-dominant position or lower, specifically being in a sub-inferior position at density D₁ (Figure 2D). Additionally, we found that the sex and number of adjacent trees of reproductive plants were influenced by the stand density (Figure S1).

3.2. Growth Traits

Density has a significant impact on the growth amount of tree height (TH), crown width (CW), and tree volume of timber (TV); sex has a highly significant effect on diameter at breast height (DBH) and TV; the interaction between density and sex has a highly significant effect only on the growth amount of CW (Figure 3). The DBH growth of female plants in D₂ and D₃ densities was significantly greater than that of plants with an unknown-sex (Figure 3A). The TH of reproduction and plants with an unknown gender in the D₁ density was significantly greater than those in other densities; in the D₂ density, the TH growth of female plants and those with an unknown sex was significantly greater than that of male plants (Figure 3B). With increasing density, the annual crown width increment of female and male individuals showed a gradual increasing trend, whereas that of individuals of unknown sex exhibited a decreasing trend (Figure 3C).The TV of male plants in the D₁ density was significantly greater than that of those in the D₄ density. Additionally, the TV of plants with an unknown sex in the D₁ density was significantly greater than that in the other three densities. Furthermore, the CW of female plants in the D₁ to D₃ densities was significantly greater than that of plants with an unknown sex (Figure 3D).

3.3. Nutritional Organ Morphology

Density has a significant effect on leaf shape, specific leaf area, the length and thickness of current years shoots, root diameter, and root tissue density; gender has a significant effect only on root diameter (Figure 4). The leaf shape of female plants in the D₁ and D₂ densities was significantly larger than that in the D₄ density, while the leaf shape of unknown-sex plants in the D₄ density was significantly larger than that in the D₁ density. (Figure 4A). The specific leaf area of male plants and unknown-sex plants in the D₄ density was significantly greater than that in the D₁ density; additionally, the specific leaf area of unknown-sex plants in the D₂ density was significantly greater than that of male plants (Figure 4B). The length of current year shoots of male plants in the D₂ density was significantly greater than that of those in the D₃ density, and that of the female and unknown-sex plants in the D₃ density was significantly greater than that of males (Figure 4C). In the D₁ density, the thickness of the current-year shoots of female, male, and unknown-sex plants was significantly greater than that of those in the D₃ density (Figure 4D). The root diameter of female and male plants in the D₁ density was significantly greater than that of those in the D₄ density; furthermore, in both the D₁ and D₄ densities, the root diameter of male plants was significantly greater than that of unknown sex individuals (Figure 4E). The specific root length of female plants in the D₂ density was significantly greater than that of those in the D₄ density, while the specific root length of unknown-sex plants in the D₄ density was significantly greater than that of those in the D₂ density (Figure 4F). The root surface area of unknown-sex plants in the D4 density was significantly greater than that of those in the D₂ density, and the root surface area of female plants in the D₃ density was significantly greater than that of male plants (Figure 4G). The root tissue density of female plants in the D₂ density was significantly greater than that of those in the D₄ density, whereas the root tissue density of male plants in the D₃ density was significantly greater than that of those in the D₄ density (Figure 4H).

3.4. Nutrition in Vegetative Organs

Density has a significant impact on leaf phosphorus and potassium content, while sex significantly affects leaf nitrogen and potassium content. In the D₃ density, the phosphorus and potassium content of leaves from female plants and unknown-sex plants was significantly greater than that of those in the D₁ density (Figure 5A–D). Density, as well as the interaction between density and gender, has a significant effect on the stoichiometry of shoots, while sex only significantly affects shoots phosphorus content. In both the D₁ and D₃ densities, the phosphorus content of shoots from female plants was significantly higher than that of male and unknown-sex plants. The phosphorus content of branches from unknown-sex plants in the D₂ density was significantly greater than that of reproductive individuals. Additionally, the phosphorus content of branches from male plants in the D₄ density was significantly higher than that of female and unknown-sex plants (Figure 5E–H). Density has a significant effect on the carbon and nitrogen content of the absorptive roots, while gender significantly affects the carbon, nitrogen, and phosphorus content of the absorptive roots. The interaction between density and sex significantly influences the carbon and phosphorus content of the absorptive roots. The potassium content of absorptive roots from female plants in the D₁ density was significantly lower than that of those in other densities, while the potassium content of absorptive roots from male plants in the D₂ density was significantly lower than that of those in the D₁ and D₄ densities. The potassium content of absorptive roots from male and unknown-sex plants in the D₁ density was significantly higher than that of female plants, whereas in the D₂ density, the potassium content of female plants was significantly higher than that of males (Figure 5I–L).

3.5. Rhizosphere Soil Physicochemical Properties

The pH of the rhizosphere soil ranged from 4.8 to 5.2, and the moisture content varied between 16% and 20.58% (Figure S2). The ammonium nitrogen content in the soil of female plants shows a trend of first increasing and then decreasing, with the maximum value occurring at D₂ density. The soil nitrate nitrogen content of reproductive individuals in low densities (D₁ and D₂) was significantly greater than that of those in high densities (D₃ and D₄, p ≤ 0.05). As density increased, the total carbon, nitrogen, and potassium content in the soil of reproductive plants and those with unknown sex all exhibited a gradual decreasing trend (p ≤ 0.05). In the D₃ density, the total phosphorus content of the rhizosphere soil in female plants was significantly greater than that of those in the D₁ density. In the D₄ density, the total phosphorus content in male plants was significantly greater than that of those in both the D₁ and D₂ densities Additionally, in the D₁ density, the total phosphorus content of plants with an unknown sex was significantly greater than that of those in the D₂ and D₃ densities (p ≤ 0.05). In the D₃ density, the rhizosphere soil nitrate nitrogen content of plants with an unknown sex was significantly greater than that of females. In the D₁ density, the total carbon content in the rhizosphere soil of reproductive plants was significantly greater than that of plants with an unknown sex, while in the D₃ density, plants with an unknown sex exhibited a significantly higher total carbon content than females. The total nitrogen content in the rhizosphere soil of reproductive plants in the D₁ density was significantly greater than that of plants with an unknown sex, whereas in the D₃ and D₄ densities, the total nitrogen content in the rhizosphere soil of plants with an unknown sex was significantly greater than that of reproductive plants. Finally, in the D₁ density, the total phosphorus content in the rhizosphere soil of plants with an unknown sex was significantly greater than that of reproductive plants (p ≤ 0.05,

Table 1. Chemical properties of rhizosphere soils in male and female individuals under different density conditions.

Density	Gender	SAN (mg·g−1)	SNN (mg·g−1)	SCC (mg·g−1)	SNC (mg·g−1)	SPC (mg·g−1)	SKC (mg·g−1)
D1	Female	3.20 ± 0.58 Ba	52.33 ± 3.49 Aa	80.14 ± 1.06 Aa	6.80 ± 0.27 Aa	0.71 ± 0.05 Bb	4.80 ± 0.27 Aa
	Male	5.09 ± 0.85 Aa	52.93 ± 2.23 Aa	76.19 ± 3.16 Aa	7.27 ± 0.30 Aa	0.71 ± 0.09 Bb	4.17 ± 0.20 Aa
	Unknown	3.38 ± 0.40 Aa	48.63 ± 3.50 Aa	61.27 ± 2.84 Ab	5.68 ± 0.19 Ab	0.98 ± 0.03 Aa	4.81 ± 0.05 Aa
D2	Female	6.20 ± 0.73 Aa	46.95 ± 2.49 Aa	62.97 ± 5.39 Ba	5.89 ± 0.52 Aa	0.90 ± 0.05 ABa	4.37 ± 0.10 ABa
	Male	6.73 ± 1.38 Aa	46.58 ± 2.78 Aa	61.92 ± 4.78 Ba	6.03 ± 0.47 Ba	0.89 ± 0.08 Ba	4.32 ± 0.09 Aa
	Unknown	3.95 ± 0.51 Aa	40.97 ± 5.21 Aa	58.66 ± 2.64 Aa	5.60 ± 0.25 Aa	0.79 ± 0.09 Ba	3.98 ± 0.23 Ba
D3	Female	4.13 ± 0.49 Ba	29.68 ± 3.13 Bb	37.21 ± 3.33 Cab	3.10 ± 0.08 Bb	0.99 ± 0.11 Aa	3.95 ± 0.10 BCa
	Male	4.68 ± 0.44 Aa	34.19 ± 4.26 Bab	31.62 ± 2.59 Cb	3.22 ± 0.18 Cb	0.92 ± 0.03 ABa	3.98 ± 0.11 ABa
	Unknown	3.83 ± 0.22 Aa	45.60 ± 4.72 Aa	45.27 ± 0.90 Ba	6.19 ± 0.37 Aa	0.75 ± 0.06 Ba	3.70 ± 0.03 Ba
D4	Female	3.94 ± 0.65 Ba	29.03 ± 5.99 Ba	34.78 ± 2.14 Ca	2.03 ± 0.07 Cc	0.84 ± 0.05 ABa	3.77 ± 0.17 Ca
	Male	4.66 ± 0.11 Aa	27.86 ± 1.52 Ba	28.83 ± 5.10 Ca	2.62 ± 0.11 Cb	1.03 ± 0.08 Aa	3.66 ± 0.14 Ba
	Unknown	3.35 ± 0.15 Aa	41.97 ± 3.51 Aa	34.78 ± 2.14 Ca	3.20 ± 0.10 Ba	0.88 ± 0.03 ABa	3.75 ± 0.24 Ba

Note: Uppercase letters indicate significant differences in the same-sex traits at different densities (p ≤ 0.05), while lowercase letters indicate significant differences between different sexes at the same density (p ≤ 0.05). SAN: Soil ammonium nitrogen content; SNN: Soil nitrate nitrogen content; SCC: Soil carbon content; SNC: Soil nitrogen content; SPC: Soil phosphorus content; SKC: Soil phosphorus content.

).

3.6. Comprehensive Analysis of Growth Traits and Soil Physicochemical Properties

For female plants, Carbon content of shoots (SN) and Phosphorus content of shoots (SP) were significantly positively correlated with Nitrogen content of leaves (LN) and Phosphorus content of leaves (LP), while SN was significantly negatively correlated with Carbon content of absorptive roots (RC). Additionally, Potassium content of shoots (SK) showed a highly significant negative correlation with some soil physicochemical properties. LP was significantly positively correlated with Potassium content of leaves (LK) and Soil phosphorus content (SPC), but significantly negatively correlated with Soil carbon content (SCC) and Soil nitrogen content (SNC). Nitrogen content of absorptive roots (RN) was significantly negatively correlated with Phosphorus content of absorptive roots (RP) and Potassium content of absorptive roots (RK), as well as with SCC and SNC. RP was significantly positively correlated with RK and showed a significant non-positive correlation with Soil water content (SWC) and SNC. RK was significantly negatively correlated with SCC. There were significant positive correlations among some soil chemical stoichiometries. Mantel test analysis indicated that the growth traits of female plants at different densities were mainly related to changes in SCC, SNC, Soil potassium content (SKC), and Soil nitrate nitrogen content (SNN) (Figure 6A). For male plants, SC was highly significantly negatively correlated with RC, while SN, SP, and SK were significantly negatively correlated with both RC and RN, as well as with SCC, SNC, and SNN at highly significant levels. LC was significantly negatively correlated with RN, SNC, and SNN, but showed a significant positive correlation with SPC. RC and RN were significantly positively correlated with some soil properties. Similarly, some soil chemical stoichiometries also showed significant positive correlations. Mantel test analysis indicated that the growth traits of male plants at different densities were mainly related to the nutrient content in branches and the changes in SCC, SNC, and SNN in the rhizosphere soil (Figure 6B).

After conducting a collinearity test for the indicators, we selected three rhizosphere soil indicators and six nutrient organ indicators for principal component analysis (PCA). Overall, the stoichiometry of the aboveground nutritional organs for both female and male plants was regulated by SWC and SPC, while the nitrogen content in absorptive roots was regulated by SNN (Figure 7). For female plants, the first two axes explained 64.5% of the overall variation. The positive axis of PC1 was primarily composed of nitrogen and phosphorus content in nutritional organs, as well as SPC, while the negative axis was composed of RN and SNN; SC was located on the negative axis of PC2. Meanwhile, female plants tended to cluster at the negative end of the PC1 axis under low densities (D₁ and D₂); however, at high densities (D₃ and D₄), they generally clustered on the positive axis of PC1 (Figure 7A). For male plants, the first two axes explained 63.7% of the overall variation. The positive axis of PC1 was mainly composed of nitrogen and phosphorus content in leaves and branches, as well as SWC and SPC, while the negative axis was also composed of RN and SNN. Additionally, RP was located on the positive axis of PC2, while SC was located on the negative axis of PC2 (Figure 7B).

4. Discussion

4.1. The Response of Spatial Structure in Male and Female Plants to Density

The spatial structure of forest trees is a fundamental element of plantation management and significantly impacts the growth processes and horizontal structures of trees [34,35]. Our study found that the competition intensity experienced by non-reproductive plants within the D₁–D₃ density range was significantly greater than that of reproductive plants. However, the competition intensity for reproductive plants within the D₄ range was significantly greater than that of those within the D₁ density (Figure 2). In addition, reproductive plants across all densities occupied a sub-dominant position, and the degree of gender mingling among reproductive plants in the D₂–D₄ density range was significantly greater than that among non-reproductive plants. This phenomenon has also been observed in other tree species, such as Populus cathayana [36,37] and alpine willows [38]. However, studies on Populus deltoides [39] and Populus cathayana [40] have suggested that male plants have greater growth space within the species. This suggests that the spatial heterogeneity of growth between male and female plants may be related to environmental factors [6] and reproductive costs [41].

As density increases, the number of same-sex plants located near female trees gradually decreases (Figure S1). This finding is consistent with the conclusions of Zhang [42] regarding F. mandshurica in natural secondary forests. Both female and male trees experience negative effects on their growth rates due to same-sex competition, with female trees being more significantly affected [42]. This indicates that female trees may require more growth resources, including adequate light and nutrients, to achieve optimal growth [43], whereas the resource demands for male trees may be weaker than those for females [44]. There are inherent reproductive differences between females and males, with the reproductive costs for females often being higher than for males [14,45]. The competition for nutrients among reproductive individuals can affect the development and reproduction of neighboring trees [7]. We speculate that reproductive trees may accelerate nutrient absorption during flowering and fruiting periods, thereby influencing the growth processes of neighboring trees [46]. It can be inferred that recognition signals may exist among individuals of different sexes within the same species, leading to differences in growth and development among individuals in the spatial structure, as well as to the phenomenon of same-sex exclusion [46].

4.2. Response of Morphological Development of Male and Female Plants to Density

Previous studies have found that male trees exhibit higher growth rates and wood accumulation capabilities compared to female trees [47]. Our research indicated that, within the D₁–D₃ density range, the wood accumulation capacity of reproductive plants was significantly greater than that of plants with an unknown sex. Furthermore, female plants possess wood accumulation abilities that are comparable to those of males (Figure 3). Although female trees allocate more resources to reproduction than males [12], our study did not find significant sex differences on growth traits at similar densities. This finding also supports the notion that female plants may adopt reproductive compensation strategies and other adaptive mechanisms to balance their reproductive investments [48].

In this study, the morphology of leaves and stems, as well as the average diameter and tissue density of roots, were primarily influenced by changes in density, with sex having a significant impact only on the average diameter of the absorptive roots (Figure 3). Changes in the morphology of leaves and stems reflect the plant’s ability to absorb and store nutrients above ground [49], while root tissue density and diameter indicate the plant’s capacity for non-structural carbohydrate accumulation in its roots [50]. When external environmental conditions change, such as during nutrient deficiency or variations in intra- or interspecific competition intensity, tree growth can be constrained [51]. An optimal stand density can enhance trees’ ability to capture light and acquire soil nutrients; however, an excessively high density can increase competition among individuals, negatively affecting their nutrient absorption capacity [26]. In this study, low density (D₁ and D₂) was found to be more conducive to promoting the morphological development of the nutritional organs of both reproductive and unknown-sex plants.

4.3. The Response of Nutrients in Reproductive Organs of Male and Female Plants and the Physicochemical Properties of Rhizosphere Soil to Density

As female and male plants transition from vegetative growth to reproductive growth, their demand for light, nutrients, and water increases during flowering and fruiting [11]. Both sexes exhibit preferences for nutrient absorption and storage strategies [52]. This study found that the leaves and absorptive roots of F. mandshurica store more nitrogen and phosphorus than the stems, while the potassium content in leaves is higher than that in both stems and absorptive roots (Figure 5). Another study on the seasonal dynamics of nutrient content in the reproductive organs of male and female Fraxinus velutina revealed that, in most months, the C and P content in female leaves was higher than in male leaves, whereas the N content was lower [53]. The sampling period for this study occurred during the peak growing season for F. mandshurica in the research area, and, by this time, the reproductive process had already concluded. Therefore, no significant differences were observed in the nutrient content of leaves and roots between females and males at the same density. In future research, we will conduct seasonal sampling to explore the dynamic changes in nutrient content in the reproductive organs of both sexes at varying densities. Regarding the soil, density significantly affects changes in nutrient content within the rhizosphere; as density increases, the overall trend is a gradual decrease in nutrient content (Table 1). Changes in stand density influence microclimatic factors, such as light, temperature, and water, which can directly affect soil conditions and nutrients availability within the forest [54].

4.4. The Interactive Mechanisms Between the Developmental Strategies of Male and Female Plants and Density

Our study found that changes in the growth traits of reproductive plants were significantly correlated with the contents of SCC, SNC, and SNN. The nitrogen and phosphorus content in the nutrient organs of both female and male individuals shows a strong correlation with the changes in nitrogen and phosphorus content in the rhizosphere soil (Figure 6 and Figure 7). One study found that gender competition patterns (intra- or intersexual) and nitrogen supply levels significantly affect the sexual dimorphism and competitive ability of Populus cathayana populations [36]. Another study on the reproductive individuals of F. mandshurica in secondary forests indicated that the radial growth rates of female and male plants were significantly influenced by soil moisture and tree size, with intra-sex competition primarily focused on nitrogen [25]. In dioecious monoculture plantations, intense gender competition and nitrogen limitations can severely hinder the growth and development of trees [55]. Additionally, we found a significant correlation between the nutrient content of the nutrient organs of female and male plants and SNN, SPC, and SWC in the rhizosphere soil (Figure 7). Existing research has indicated that gender effects may lead to changes in rhizosphere fungal communities and soil properties [56], and stand density also has a considerable impact on rhizosphere soil nutrients [57]. Our results show that although stand densities vary, the LP of female individuals was significantly correlated with SPC, while the LP of male individuals was significantly positively correlated with SWC. These relationships suggest that female and male individuals of F. mandshurica exhibit both common and dimorphic developmental characteristics in response to changes in density.

In this study, F. mandshurica were in the early stage of sex development, and some trees in the forest had not yet entered reproductive growth. However, for woody trees with long lifespans, it may take over a decade or even decades for all individuals to enter reproductive growth. Therefore, investigations during the early stage of sex development in forest stands are particularly important.

5. Conclusions

This study reveals the impact of density on the growth space size and composition of male and female trees in F. mandshurica plantations, as well as on the morphology and nutrient content of their nutrient organs and the properties of rhizosphere soil, from both aboveground and belowground perspectives. Our findings indicate that, as density increases, the competition intensity experienced by both female and male plants within the spatial structure also intensifies, while the number of neighboring individuals of the opposite gender decreases. Furthermore, density significantly affects the annual growth increment in tree height, with additional analysis showing that it primarily influences the biomass accumulation capacity of male and unknown-sex individuals. Both female and male plants were more susceptible to changes in density regarding specific leaf area, shoots thickness, absorptive root diameter, and root tissue density. Density was a major influencing factor for variations in nitrogen and phosphorus content in nutrient organs, and it was closely related to water content, ammonium nitrogen, and total phosphorus levels in the rhizosphere soil. Monitoring the reproductive development of trees was crucial when managing F. mandshurica plantations, particularly ensuring that reproductive individuals have sufficient growing space. These results underscore the importance of considering density and spatial structure in the management of dioecious tree species to promote healthy growth and successful reproduction. Future research will involve long-term observation of this plot. By further investigating other potential factors, such as climate change, soil microbial, and hormone levels, the general applicability of these conclusions will be validated. The findings of this study offer valuable theoretical insights and practical guidance for the management and cultivation of plantations consisting of other dioecious tree species. Specifically, the results can inform strategies related to sex differentiation mechanisms, growth regulation, and silvicultural practices, thereby contributing to the optimization of forest productivity, sustainability, and genetic diversity in analogous artificial forest systems.