**1. Introduction**

Leaf nitrogen (N) and phosphorus (P) are the two most limiting elements of terrestrial vegetation relating to plant growth, development, and reproduction [1–3] because N is the key component of proteins that play pivotal roles in plant photosynthesis as enzymes, whereas P is critical to the formation of NADPH, ATP, and ribosomal RNA in the process of protein synthesis [4,5]. Furthermore, the critical leaf N:P ratio has been widely used to diagnose the type of nutrient limitations to plant productivity [6–8]. Leaf N and P concentration and allocation have been found to vary along environment gradients; although numerous studies have focused on their latitudinal and altitudinal patterns [9–16], it has rarely been studied how they change along the slope aspect gradient. Investigations on leaf N:P stoichiometry across slope aspects can provide applicable guidance in the refinement of grassland management and conservation based on topography at the local scale.

Leaf N and P availability also shows important functional significance for community composition [6]. In particular, relationships between plant N:P ratios and species richness are of particular interest in the context of biodiversity conservation and anthropogenic activities. Many studies have found the N:P ratio to be correlated with the richness and composition of species [17–19]. The coexistence of species was suggested to be possibly facilitated by P limitation, since the competition for P with lower mobility is weaker than the

**Citation:** Li, X.; Hu, Y.; Zhang, R.; Zhao, X.; Qian, C. Linking Leaf N:P Stoichiometry to Species Richness and Composition along a Slope Aspect Gradient in the Eastern Tibetan Meadows. *Diversity* **2022**, *14*, 245. https://doi.org/10.3390/ d14040245

Academic Editors: Lin Zhang, Jinniu Wang and Michael Wink

Received: 5 March 2022 Accepted: 26 March 2022 Published: 27 March 2022

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competition for resources of high mobility in soil [17,20] and is facilitated by colimitation because differential nutrient limitations may reduce interspecific competition [21,22] but could also be weakened by the P limitation possibly resulting from P deficiency or nutrient imbalance [6] or the disproportionate increase in dominant clonal graminoids [23]. Hence, different correlations of species richness with plant N and P stoichiometry have been reported [17,18,24–26]. Moreover, a high leaf N:P ratio was expected to be associated with more graminoids and fewer forbs in vegetation [23]. However, most of these studies were indirectly demonstrated in experiments with nitrogen fertilization or deposition, and direct evidence is rare along natural environmental gradients. The slope aspect gradient significantly contributed to the heterogeneity of vegetation in mountain areas and thus provided an ideal platform to explore the relationships between leaf N/P stoichiometry and community structure.

Typically, south-facing slopes in the northern hemisphere (i.e., equator-facing slopes) are hot and dry, as the equator-facing orientation is associated with the strongest solar irradiation, whereas north-facing slopes (i.e., polar-facing slopes) are wet and cold, resulting from the lowest solar irradiations; western- or eastern-facing slopes are intermediate in terms of these aspects. Slope aspect substantially contributes to the heterogeneity of vegetation [27–32], possibly owing to the substantial microenvironmental changes, such as solar irradiance, soil moisture and temperature, and soil nutrients [33–36]. However, it is still not clear how vegetation heterogeneity, including species richness and composition, is associated with leaf N:P stoichiometry. Therefore, in the current study, based on a slope aspect gradient in the Tibetan meadow, we mainly aimed to explore the following questions: (1) How do leaf [N] and [P] and the N:P ratio vary in different slope aspects? (2) How do species richness and composition change with slope aspect? (3) How does leaf N:P stoichiometry correlate with species richness and composition along the slope aspect gradient?

### **2. Materials and Methods**

### *2.1. Study Sites*

Our study was conducted in an alpine meadow in the eastern part of the Tibetan Plateau in China. The Research Station of Alpine Meadow and Wetland Ecosystems of Lanzhou University has locations at two elevations: 2960 m and 3650 m (Figure 1A). Data were collected at these two sites: Hezuo (34◦44 N,102◦53 E) and Maqu (33◦39 N,101◦53 E), with the vegetation landscape shown in Figure 1B. We also recorded the precipitation and temperature of these two sites during the period of 1981–2017 according to geographical coordinates using the climate dataset provided by National Tibetan Plateau Data Center (http://data.tpdc.ac.cn (accessed on 10 May 2021)). The mean annual precipitation in Hezuo and Maqu was 570 mm and 690 mm, and the mean annual temperature was around 4 ◦C and 2 ◦C, respectively. The monthly mean precipitation and monthly mean, maximum, and minimum temperatures are shown in Figure 1C. Details about the study site can also be found in our previous publications [36,37].

### *2.2. Leaf N and P Concentration Measurements*

In August of 2008, during the peak growing season,a5m × 5 m plot was established on each of the south-, west-, and north-facing slopes (i.e., SFS, WFS, and NFS) based on the shape of the hill and the ability to collect leaf samples at each site. At each plot, mature and healthy leaf samples were collected from 3–5 individuals of the dominant species. Across the six plots, 80 observations were collected in total, with 41 observations of 25 species in Hezuo and 39 observations of 20 species in Maqu. The measured species in each site, with their average leaf N and P content per unit of mass (hereafter leaf [N] and [P]), are shown in Table 1. All the species were simply classified into three plant functional groups (PFGs): graminoids (Poaceae and Cyperaceae), non-legume forbs (forbs), and legumes. However, in two plots, dwarf shrub was also found on the NFS.

**Figure 1.** Site location in the Tibetan Plateau (Panel **A**), the vegetation landscape (Panel **B**), and key climate factors (Panel **C**) of the two study sites (Hezuo and Maqu). Tmean, Tmax, and Tmin represent the monthly mean, maximum, and minimum temperature, respectively.

After drying for 48 h at 70 ◦C, we ground the dry leaves to powder using a mortar. A total of 0.2 g of leaf powder was digested with H2SO4-H2O2. Digested solution was used to determine leaf [N] with a VAPODEST 40 programmable distillation system (Gerhardt, Germany), and leaf [P] by the vanadium-ammonium molybdate colorimetric method [38].
