Interspecific differences in nitrogen form acquisition strategies contribute to species dominance

Plant’s ability to use prevalent or less prevalent soil nitrogen (N) forms may affect their dominance within vegetation types, and these partitioning-driven changes in dominance may facilitate species co-existence. However, the mechanisms underlying these processes remain unclear, particularly given the strong influence of altitude on soil N forms, which in turn affects plant N acquisition strategy. In this study, we first determined the effects of preference and plasticity in N form uptake on partitioning of soil N forms and species dominance, and then assessed the relative importance of these two N form use strategies for 19 dominant and non-dominant species in three vegetation types along an altitudinal gradient on Changbai Mountain, northeast China. To achieve this, we measured dominance, the contents of different N forms in rhizosphere soils, their proportional contributions to leaf N, and N form uptake preference and plasticity for these 19 species. Our results show significant interspecific differences in the proportional contributions of different soil N forms to leaf N within all three vegetation types, providing a novel mechanism underlying niche differentiation among plants. Species dominance was positively associated with the proportional contributions of soil dissolved organic N (the most prevalent N form) and the main inorganic N form to leaf N, while negatively with that of the subordinate inorganic N. These associations were not altered by the altitude-driven changes in the absolute and proportional contents of different soil N forms, suggesting a potentially widespread phenomenon. Both preference and plasticity in N form uptake contributed to the proportional contributions of different N forms to leaf N, and therefore to species dominance and co-existence within vegetation types. Furthermore, N form preference was more critical for non-dominant relative to dominant species and at high relative to low altitude, while N form uptake plasticity was more important for dominant species and at low altitude. Our study provides robust evidence for the interspecific niche differentiation in N form uptake, contributing to species dominance and co-existence within vegetation types, and reveals the mechanisms (plasticity and preference) underlying the association between species dominance and the uptakes of different N forms.

Methods
Study sites

This study was conducted on the north slope of Changbai Mountain, Jilin Province, northeast China (41°23′ – 42°36′ N; 126°55′ – 129°00′ E). This region has a typical continental temperate monsoon climate, with long cold winters and short warm summers. From the lower part of the mountain at 740 m to the summit at 2691 m, the mean annual temperature decreases from 2.8 to –7.3oC and the mean annual precipitation increases from 750 to 1340 mm (He et al., 2005).

Korean pine and broadleaved mixed forest (KPBL), Erman’s birch forest (EB), and alpine tundra (AT) were selected as study plant communities based on their prevalent forms of soil N. In KPBL, dissolved inorganic N (DIN) accounts for a large proportion of total dissolved soil N (TDN), and NO3- is the prevailing inorganic N form; in EB, DIN also accounts for a large proportion of soil TDN, but NH4+ is the predominant inorganic N form; in AT, however, DON is the principal soil N form (CERN; http://www.cerndata.ac.cn/). Similar altitudinal changes in N forms were also found in our study (Appendix S1: Figure S1). The main properties of the three vegetation types are summarized in Appendix S1: Table S1. The sample plots are located in a national nature reserve, and thus human interference is negligible.

Study species

In mid-July of 2018, three 30 × 30 m plots (> 20 m apart from one another) were randomly established in EB and AT, respectively. First, we investigated the background information such as altitude, slope degree and vegetation cover in each plot. Second, all woody plants [diameter at breast height (DBH) ≥ 1 cm] were identified. Third, we measured the number, DBH, basal area, crown width, and height of each individual of each tree species, and the number, height and cover of each individual of each shrub species in each plot. Finally, we calculated relative density (RDy) and relative frequency (RF) for the tree and shrub species, relative dominance (RDe) for the tree species, relative cover (RC) for the shrub species, and then the importance values (IV) for the tree and shrub species in each plot based on Zhang et al. (1995) and Asigbaase et al. (2019). The importance value was used to quantified species dominance in our study.

        IV of tree = (RDy + RF + RDe) / 300……. ………………………………….……………(1)

        IV of shrub = (RDy + RF + RC) / 300………………………………………………..……(2)

        RDy = number of individuals of the species / total number of individuals of all species × 100……………………………..………………………………………………………………(3)

        RF = frequency of the species / sum frequency of all species × 100……………………..(4)

        RDe = basal area of the tree species / total basal area of all tree species × 100…………..(5)

        RC = cover of the shrub species / total cover of all shrub species × 100……………..…..(6)

For KPBL, three 30 × 30 m plots were randomly established in a 25-ha KPBL permanent plot. In the permanent plot, only 17 woody plant species had more than 10 individuals per hectare, which accounted for more than 95% of the individuals of all woody plants (Hao et al., 2008). Our studied species were selected from these 17 species using the following criteria: individuals > 3 in each plot; and only one species was randomly selected when more than one from the same family had similar numbers of individuals in each plot. Ultimately, 10 species were selected, and their importance values were calculated using the methods described above. These species together accounted for more than 80% of the total aboveground biomass (Li et al., 1981).

In total, 19 woody plant species were studied, and only one occurred in more than one vegetation types, i.e., Rhododendron aureum in both EB and AT (Appendix S1: Table S2). The species were divided into dominant (≥ 3) and non-dominant (< 3) according to their importance values (Avolio et al., 2019). The numbers of the dominant and non-dominant species are 6 and 4, 3 and 3, and 2 and 1 in KPBL, EB, and AT, respectively (Appendix S1: Table S2).

Leaf and soil sampling, measurements and calculations

In August of 2018, 10 fully developed, intact and healthy leaves or bunches of current-year needles on the south side of the upper canopy were randomly collected from each of the three sampling individuals for each studied species in each plot. The leaves or needles were collected using tree pruners when the sampling individuals were short. For high trees, we climbed up to collect the leaves and needles. The leaves or needles from the same species in each of the three plots were mixed as one sample. This sampling procedure ensured a representative and homogeneous sample, reducing potential effects of intraspecific differences within-plot.

Rhizosphere soils were also collected from the sample individuals mentioned above. We first carefully removed the litter and humus layers from the soil surface at four sides (east, south, west and north) around each sample tree (≈ 1 m from the trunk). Then, fine roots (diameter < 2 mm) were dug out at each side, and rhizosphere soils were collected using a hand-shaking method (Guan et al., 2023). Soils from the same species in each plot were thoroughly mixed as one sample. The soils were immediately put into an ice box, transported to the laboratory of the Research Station of Changbai Mountain Forest Ecosystems, Chinese Academy of Sciences, passed through a 2 mm sieve, and stored at 4oC until measurements.

Leaf total N concentration and δ15N were measured using an element analyzer-stable isotopic mass spectrometer (Flash EA 1112 HT-Delta V Advantages, Thermo Fisher Scientific, USA). The contents of DIN (NH4+ and NO3-), DON, and TDN in rhizosphere soil of each sample species were measured using the standard method commonly used in literature (Guan et al., 2023; Zhang et al., 2018). Soil water content was measured using the oven-drying method. The δ15N values of soil NH4+, NO3-, and TDN (δ15NNH4+, δ15NNO3-, and δ15NTDN) were measured using the chemical method (Liu et al., 2014), azide method (Tu et al., 2016), and high-temperature catalytic oxidation techniques performed with a total organic carbon analyzer-stable isotope ratio mass spectrometer (Iso TOC cube-isoprime100, Elementar, Germany), respectively. The δ15N value of DON (δ15NDON) was calculated as the mass-weighted difference between δ15NTDN versus δ15NNH4+ and δ15NNO3-.

The measurement data mentioned above were used to estimate the proportional contributions of soil NO3-, NH4+, and DON to leaf N (fNO3-, fNH4+, and fDON; %) (Houlton et al., 2007; Stock et al., 2018). The effects of possible isotope fractionation during within-plant N allocation and/or N transport from mycorrhizal fungi to host plants were taken into account (Houlton et al., 2007; Zhu et al., 2019). Plant preferential uptake for soil NO3-, NH4,+ and DON (βNO3-, βNH4,+ and βDON) were estimated by comparing their proportional contribution to leaf N and their relative contents in soil (Guan et al., 2023; Zhang et al., 2018). To estimate plasticity in N form uptake, percentage similarity between plant uptake pattern of different N forms and their availability pattern in rhizosphere soil (percentage similarity) was determined for each species in each plot based on McKane et al. (2002) and Guan et al. (2023).

For detailed measurement and calculation methods please see Appendix S1: Section S1, and mean value of three plots was reported for each variable in the results section.