The study selected the natural populations of the Sea-buckthorn genus, which are primarily distributed across the Loess Plateau and the Qinghai-Tibet Plateau. The research focuses on three dominant species regionally distributed in China: Chinese sea-buckthorn, Tibetan sea-buckthorn, and Coastal sea-buckthorn. A total of 110 sampling sites were identified ( Figure 1 ). Of these, 74 natural populations were for Chinese sea-buckthorn in the Loess Plateau and eastern margin of Qinghai-Tibet Plateau, covering the provinces of Hebei, Inner Mongolia, Shanxi, Shaanxi, Ningxia, Gansu and Qinghai (34°-40°N, 100°-114°E; 1024–3209 m a.s.l.). The 25 natural populations sampling sites of sea-buckthorn in Tibet were largely situated in the southeastern Qinghai-Tibet Plateau, spanning four provinces: Gansu, Qinghai, Tibet, and Sichuan (29°-38°N, 88°-103°E; 2869–4860 m a.s.l.). According to the native range of sea-buckthorn in the Loess Plateau and Qinghai-Tibet Plateau, 11 natural populations were selected as sampling sites. Average annual temperature (MAT) ranged from –5.87 to 4.53°C, and average annual precipitation (MAP) ranged from 222–766 mm ( Figure 1 ).

In each sampling site, a plot was set up (n = 110), in which sea-buckthorn leaves with good development, uniform growth, and lacking any disease symptoms were selected. For each species, 30 individual mature leaves were collected from different directions and mixed into a single sample, with three such leaf composite samples obtained per plot (330 leaf samples in total). Each sample was put into a labeled envelope and taken to the laboratory. There, all leaf samples were dried at 65°C to constant weight, crushed, and screened through 100-mesh to determine their respective N and P concentrations. The leaf N concentration was measured using the Kjeldahl method, while the leaf P concentration was measured with the molybdenum-antimony resistance colorimetric method (Jones, 2001).

The climate data used in this study came from the WorldClim website (http://worldclim.org). This dataset provides global average monthly meteorological data, at 1km×1km resolution, for 30 recent years. Monthly data were obtained by inputting the coordinates of each sampling site (in Figure 1 ), for which the annual average data is then calculated, with respect to three sets of factors: energy, moisture, and terrain. The energy factors were the maximum temperature (MAXT), minimum temperature (MINT), average annual radiation (Ssrad), warmth index (WI). Among water factors were the potential evapotranspiration (PET), and humidity index (HI), average annual rainfall (MAP), and Vapor Pressure (Vapr) were considered here. Topographic factors included wind, altitude, aspect, and slope. ArcGIS 10.2 software was used to extract meteorological data for each sampled plot, according its latitude and longitude. Potential evapotranspiration (PET), warmth index (WI), and humidity index (HI) were calculated according to the methods of Zhang (1989) and Zhang et al. (2017).

In processing the data Excel 2019 software was used for preliminary data sorting, after which SPSS 25 software was used for formal data analyses. One-way analysis of variance (ANOVA) was used to compare the differences in N, P, and N/P response variables among the three sea-buckthorn species, for which Duncan’s test were used to carry out multiple post-hoc comparisons, with statistical significance set to α = 0.05. To investigate the spatial pattern and distribution relationship of plant leaf stoichiometry, linear regression analysis was employed. The R software package UpSetR was employed for the graphical analysis of the intersection of stratified visual sets (Conway et al., 2017). This approach facilitated the identification of key environmental factors influencing nitrogen (N) and phosphorus (P) concentrations in sea buckthorn, as well as the N/P ratio. The relationship between N and P can be described by a mathematical equation, y = βxα. The linear form is log (y) = log (β) + αlog (x), x and y determine whether the relationship is isometric (α = 1.0) or allometric (α > 1.0 or α < 1.0). Standardized major axis regression (SMA) was implemented using the ‘SMART’ package in R2.0 (Warton et al., 2010). Finally, redundancy analysis (RDA) analysis was performed using Canoco 5 software, to verify the species substitution distribution according to the spatially distributed stoichiometric characteristics of sea-buckthorn leaves along the environmental axes. Figures were drawn in the R v4.3.2 platform, Origin Pro 2021(Origin Lab, USA), and Canoco 5.

As Figure 2 shows, for N, P, and N/P ratio of the sea-buckthorn were normally distributed and their skewness values were always less than 1 (Kolmogorov-Smirnov test, P > 0.05). The CV (coefficient of variation) values of N, P, and N/P respectively were 15.4%, 23.48%, and 24.04%. Among the three sea-buckthorn, the leaf N and P concentrations were higher in Chinese sea-buckthorn than Coastal sea-buckthorn, being lowest in Tibetan sea-buckthorn. However, the N/P ratio was highest in leaves of Tibetan sea-buckthorn ( Table 1 ).

The leaf N concentration of sea-buckthorn was positively correlated with longitude, and latitude (P > 0.05). Their leaf P concentration was positively correlated with longitude (P < 0.001), while not significantly correlated with latitude (P > 0.05). The N/P ratio was significantly positively correlated with latitude (P < 0.001), albeit not significantly correlated with longitude (P > 0.05) ( Figure 3 ).

The N concentration, and N/P ratio of Chinese sea-buckthorn and Coastal sea-buckthorn exhibited significant increases with rising latitude (P < 0.01). However, no significant variation was observed in the N/P ratio of Tibetan sea-buckthorn alone (P > 0.05). Additionally, the N concentration of both Chinese sea-buckthorn and Tibetan sea-buckthorn showed significant positive correlations with longitude (P < 0.01), ( Figure 4 ).

The standardised major axis (SMA) regression demonstrated Tibetan sea-buckthorn the lack of a 1:1 correspondence between N and P, but there was nonetheless a significant allometric relationship (P < 0.01), with a slope of 2.73 (95%CI: 2.15, 3.46); Chinese sea-buckthorn species, and Coastal sea-buckthorn species between N and P no significant allometric relationship (P > 0.05) ( Figure 5 ).

To further investigate the influencing factors on the stoichiometry of sea-buckthorn leaves, we employed hierarchical segmentation to systematically prioritize and rank these factors. UpSet analysis revealed that PET and altitude were the predominant factors influencing the N concentration in sea-buckthorn, followed by Ssrad ( Figure 6A ). The primary factors affecting P concentration were slope and HI, with PET being secondary ( Figure 6B ). Additionally, the N/P ratio was influenced by Ssrad, MAP, and wind ( Figure 6C ). Overall, water-related factors were identified as the major drivers of N and P concentrations (21.63% and 32.68%, respectively), while energy-related factors predominantly affected the N/P ratio (42.94%).

The redundancy analysis confirmed that environmental factors significantly influenced the stoichiometric characteristics of sea-buckthorn leaves, for which 40.6% of their total variance was explained by the two first axes ( Figure 7 ). Evidently, N was negatively correlated with potential evapotranspiration and elevation, while P was positively correlated with minimum temperature, maximum temperature, water vapor pressure, and the warmth index. Importantly, the environmental distribution of the three species (polygons in Figure 7 ) shifted towards the lower right; this being opposite to the direction of temperature increase, and also opposite to the direction of their leaf N and P concentrations increase. Hence, these results indicated that, as the temperature declined, a species shift occurred on the landscape, from Chinese sea-buckthorn to Tibetan sea-buckthorn. Moreover, there was some overlap detected in the leaf stoichiometric distribution of Coastal sea-buckthorn and Tibet sea-buckthorn, albeit the latter’s distribution was more extensive. To sum up, sea-buckthorn species with lower leaf N and P concentrations seemed more adaptable to a low-temperature environment, leading to the observed landscape pattern of plant species substitution.

All sea-buckthorn species are capable of nitrogen fixation (Zhang et al., 2024), which means these plants can fix N in the atmosphere via rhizobium symbiosis to access extra N sources (Zhu et al., 2023; Xiao et al., 2024). Using our study’s results, we find that the leaf N concentration of sea-buckthorn leaves exceeds that of shrubs, and other non-legumes in China, and terrestrial plants in its northwest alpine region, in addition to plants globally ( Table 2 ). Similarly, the leaf P concentration of sea-buckthorn surpassed that of non-legumes, and terrestrial leaves in China, being slightly higher than for plants globally, though it was not nearly as high as in China’s northwest alpine shrub leaves ( Table 2 ). The higher N concentration of sea-buckthorns could be due to the life history of sea-buckthorn, namely the N-fixing ability of these plants, endowing them with a richer source of N for their growth and development. Plant habitat and life form will also affect the stoichiometric characteristics of leaves (Lu et al., 2023). Some studies have found that, in order to adapt to a short growing season and cold environment, plant species in alpine regions tend to have short leaf life-span and high growth rate, and are able to store more lipid P in their leaves to resist cold (Han et al., 2011; van Ommen Kloeke et al., 2012). In addition, the leaf P concentration of Chinese plants has been reported to steadily increase when moving from a humid to arid climate (Li et al., 2014). Consistent with that generalization, the P concentration of sea-buckthorn genusleaves in this study is lower than those of alpine shrubs in northwest China. The N/P ratio threshold of plant growth limited by was 14 and 16, respectively. In other words, at an N/P ratio is less than 14, plant growth at community level should be restricted chiefly by N; conversely, at an N/P ratio exceeds 16, plant growth is instead limited by P. However, when the N/P ratio is between 14 and 16, both N and P is limiting to plants (Sardans et al., 2021). According to our results, sea-buckthorn plants are therefore constrained by P limited. Consistent with this inference, other research has found that N-fixing plants are susceptible to P limitation in different ecosystems (Toro et al., 2023). This is in alignment with the findings reported in the study.

Leaf stoichiometry reflects nutrient limitations in ecosystems and plant adaptations to climate change, and geographical changes of plant nutrients are undoubtedly related to climate (Freschet et al., 2018). In the present study, the N concentration increases as the longitude, and latitude increase, while the P concentration increases as longitude increase, yet both N and P concentration s decrease with rising altitude and N/P ratio increases with latitude ( Figure 3A–E ). These findings provide compelling evidence for spatial heterogeneity in the nutrient allocation strategies of sea-buckthorn species, reflecting their unique adaptation to differing environmental conditions. Sun et al. (2017) found that Tamarix Lour P concentration decreased with increasing latitude. In a later study, however, Hu et al. (2017) reported that N concentration in reed leaves decreased with increasing latitude; Wu et al. (2012) found that N and N/P in Quercus leaves decreased with increasing latitude, while its leaf P increased with increasing latitude. These discrepancies between those studies may reflect differences between plant genera sampled and complex interactions of environmental factors. It is a well-known fact that latitudinal zonation of vegetation is mainly controlled by temperature, which has a monumental influence on plant growth and metabolic processes (Hu et al., 2017). Higher temperatures are generally beneficial to plant growth, promoting nutrient absorption and metabolic activity, which helps to augment the elemental concentration in body of plants (Heckathorn et al., 2020). Further, the ability of sea-buckthorn plants to fix N is affected by temperature. Since the nitrogen source provided by surface rhizobia is affected by temperature, when this rises it may further enhance their ability to obtain N for use by plants. This could explain why, with an increasing latitude and temperature, the leaf concentration of N and P also correspondingly increases.

Our study revealed that the key factors influencing the leaf N concentration in sea-buckthorn were potential evapotranspiration and altitude, while the slope and humidity index were the primary factors affecting P concentration. Additionally, N/P ratios were predominantly influenced by annual mean solar radiation and mean annual precipitation ( Figure 6 ). This is likely due to the fact that sea-buckthorn plants are predominantly distributed in regions with an average annual rainfall ranging from 400–700 mm. Potential evapotranspiration, as a critical component of the water cycle, interacts with precipitation to determine regional aridity or humidity. The synchronization of precipitation and thermal conditions in these regions creates a more favorable environment for plant growth, thereby shaping dominant tree species and influencing leaf stoichiometric characteristics (Milly and Dunne, 2016). Altitude acts as a key variable affecting vegetation growth by modulating temperature, precipitation, and humidity, which in turn influence the stoichiometry of plant leaves (Wang et al., 2021). For instance, Zhang et al. (2021) demonstrated that plant N concentration exhibited a decreasing trend along the altitudinal gradient. Zhao et al. (2014) reported a significant reduction in the overall N concentration of leaves as altitude increased. Additionally, several studies have indicated that low temperatures in high-altitude regions impose constraints on plant growth. With increasing latitude, both temperature and humidity tend to decline gradually. Certain plant species with limited tolerance may either retreat from the community due to unsuitable habitat conditions or be excluded as a result of their insufficient competitiveness, thereby influencing the levels of N and P within the ecosystem (Bergholz et al., 2017; Heckathorn et al., 2020). The slope of influences the structure and dynamics of vegetation (Sanders and Rahbek, 2012). Previous studies have demonstrated that slope and altitude affect species composition by altering habitat temperature, and humidity, as well as the spatial redistribution of solar radiation and precipitation. Additionally, leaf stoichiometric characteristics vary among different species. Slope and elevation also play a critical role in shaping the variations in N and P concentrations within plants (Cheng et al., 2023). There is a certain relationship between the humidity index and precipitation, which is crucial for plant growth. Adequate precipitation generally increases the of N and P concentrations, thereby enhancing plant growth and nutrient absorption. Moreover, high humidity promotes plant transpiration, which in turn affects leaf stoichiometry.

Collectively, water factor, topographic factor, and energy factor significantly influence the stoichiometric characteristics of sea-buckthorn leaves. Acting as the central driving force for ecosystem function, biogeochemical cycles, and life activities, water plays a pivotal role in regulating N and P element concentrations in sea-buckthorn leaves by governing energy distribution, material transport, and biological adaptation mechanisms. These findings suggest that sea-buckthorn plants adjust their nutrient allocation strategies to adapt to environmental changes.

In the present study, the species N and P concentrations were ranked as follows: Chinese sea-buckthorn > Coastal sea-buckthorn> Tibetan sea-buckthorn, but reversed for their N/P ratios (16.16, 17.51, and 17.85, respectively; Table 1 ). According to the vegetation survey, the geographical distribution of sea-buckthorn encompasses temperate grassland area and the alpine meadow area located in the Loess Plateau and the eastern margin of the Qinghai-Tibet Plateau ( Figure 1 ). Some studies have found that the N concentration and N/P ratio in leaves of the Loess Plateau are significantly higher than those of Chinese flora and global flora (Zheng and Shangguan, 2007). In this study, Chinese sea-buckthorn is located in the eastern margin of the Loess Plateau and the Qinghai-Tibet Plateau, so it has higher nitrogen concentration than either Tibetan sea-buckthorn or Coastal sea-buckthorn. Since all species of sea-buckthorn are N-fixers, and low temperature will affect the growth and nutrient absorption of plants, a decline N-fixation function is expected, so that the leaf N concentration of Tibetan sea-buckthorn is lower than that of Chinese sea-buckthorn and Coastal sea-buckthorn. Wang et al. (2008) investigated soil P reservoirs in China, finding more available P in the Loess Plateau than the Qinghai-Tibet Plateau, while the latter’s available P was lower than that in other regions. It could well be that where the habitats of conspecific plants differ, their plant relative demands for N and P in these local environments diverges, and their energy supply is also different; altogether, this could ultimately lead to a shift in the ecological niche (Pastore et al., 2021). It has been reported that as plant growth rates increase along with higher concentrations of N and P concentrations, the N/P ratio tends to decrease (Delgado-Baquerizo et al., 2016). Chinese sea-buckthorn and rib sea buckthorn, which are found in the transition zone between the Loess Plateau and the eastern margin of the Tibetan Plateau, exhibit higher concentrations of N and P compared to Tibetan sea-buckthorn. Consequently, the levels of N and P are relatively lower in Tibetan sea-buckthorn, which is native to the Qinghai-Tibet Plateau, resulting in a higher N/P ratio compared to the other two species of sea-buckthorn.

This study also found that the N and P concentrations of the three sea-buckthorn species showed an allometric growth pattern, and there was no significant difference in N, P distribution between Chinese sea-buckthorn and Coastal sea-buckthorn, while there was a significant difference in N, and P distribution between Tibetan sea-buckthorn ( Figure 5 ). This finding aligns with the research conducted by Wang et al. (2015) on plants in the arid saline-alkali environment of Northwest China. They discovered a robust allometric relationship between N and P in leaves. However, the allometric index of Tibetan sea-buckthorn in this study differed slightly from the 3/4 index reported by Reich et al. (2010). This suggests that, relative to the P concentration in Tibetan sea-buckthorn leaves, a greater allocation of P concentration occurred. This finding implies that the growth conditions of the plants have undergone significant changes due to the increased nutrient concentration. The reason for this phenomenon may be attributed to the fact that as the temperature decreases, plants require a higher P concentration to sustain growth. Additionally, an increased P concentration can enhance the plant’s cold resistance, thereby enabling it to adapt more effectively to the changing environment. Our study revealed that the allometric exponent of Chinese sea-buckthorn, Coastal sea-buckthorn, and Tibetan sea-buckthorn exhibited a sequential increase. This finding aligns with prior studies, indicating that under more favorable climate conditions, a higher P concentration of accelerates the conversion and metabolic growth rate of plant matter, thereby resulting in a lower allometric exponent (Guo et al., 2020).

Some studies have also demonstrated that environmental factors directly influence the N and P concentrations of leaves, thereby driving intraspecific changes. Species characterized by high growth rates and strong competitive abilities tend to dominate ecosystems. Owing to variations in ecological strategies, different species exhibit distinct elemental compositions and stoichiometric characteristics, which contribute to the geographical patterns of leaf nutrient distribution via environmental filtering. This process ultimately establishes unique ecological niches for each species (Tian et al., 2024). Redundancy analysis ( Figure 7 ) revealed that with the variation in temperature, the geographical shift of species distribution from the Loess Plateau to the Tibetan Plateau, Tibetan sea-buckthorn exhibited reduced competitiveness compared to the other two sea-buckthorn species in a high-nitrogen environment. Consequently, Tibetan sea-buckthorn serves as a transitional zone between Chinese sea-buckthorn and Coastal sea-buckthorn. The gradual replacement of Chinese sea-buckthorn, which has a higher P concentration in its leaves, by Coastal sea-buckthorn and Tibetan sea-buckthorn.

This substitution reflects the plant’s adaptation to its environment, both of which exhibit lower leaf P concentrations, demonstrates plant adaptation to environmental conditions. This phenomenon aligns with the regarding species composition and biogeochemical niche hypothesis. These changes in plant stoichiometric characteristics ultimately resulted in the establishment of a relatively stable survival strategy for various sea-buckthorn species across different environmental gradients. This, to a certain extent, reflected the differentiation of sea-buckthorn species as a group, enabling their adaptation and response to environmental conditions, and eventually leading to a niche-driven species distribution pattern. These findings for sea-buckthorn echoes those of Sardans et al. (2021) emphasizing the fundamental role of plant stoichiometric characteristics in determining both species distribution and ecosystem function.