1. Introduction
The fundamental principle underlying life-history theory revolves around the notion of trade-offs and the allocation of scarce resources to either reproduction or growth and maintenance necessary for survival [
1]. Furthermore, a considerable portion of perennial species demonstrate the ability to undergo multiple cycles of growth and reproduction, enabling them to reproduce across multiple seasons. However, it is imperative to recognize that there exists notable year-to-year variability in the seed production of perennial herbs in response to the prevailing growth conditions [
1,
2]. Plants exhibit enhanced seed production when subjected to favorable years, which encompass optimal soil moisture and nutrient levels as well as suitable weather conditions [
3,
4]. In light of unfavorable years, there is a decrease in the overall yield of seeds [
1,
3]. Thus, there is a diverse range of interannual variation in seed production that is associated with different growth years.
The global distribution of resources is undergoing substantial changes due to the increasing levels of carbon dioxide (CO
2), deposition of nutrients, and alterations in land use. Predictive models indicate that multiple factors associated with global change have the potential to modify interannual variation in seed output [
3]. The introduction of nitrogen into the soil has a significant influence on the nutrient cycle, particularly in relation to the plant's ability to obtain readily available resources [
5,
6]. In theory, the introduction of nitrogen presents a challenge in allocating resources between growth and reproduction, thereby influencing the annual fluctuations in the seed production capacity of perennial plants. As a result, this phenomenon leads to an increase in their overall reproductive efficiency. Nonetheless, there is a notable deficiency in our understanding of the effects of nitrogen supply on the interannual variations in seed production patterns of perennial plant.
The impact of nitrogen on the annual fluctuations in reproductive allocation is regulated by its influence on the composition of soil nutrients [
1,
3]. During the early stages of development, plants that are grown in adequate-N habitats allocate a higher proportion of their resources towards the growth and development of their vegetative organs [
7,
8]. Subsequently, plants demonstrate an increased allocation of biomass towards their reproductive structures, resulting in an amplified production of flowers [
9,
10]. Additionally, the influence of nitrogen is a significant factor in the annual fluctuations of plant biomass, as it affects the storage of carbohydrates and the distribution of carbon resources [
11,
12]. This phenomenon possesses the capacity to induce annual variations in seed production. Additionally, the introduction of nitrogen has the potential to impact both the flower production and the nectar secretion [
13,
14,
15]. The alterations in floral traits have the potential to influence the pollinator foraging behavior, resulting in changes to the reproductive output of self-incompatibility species [
9,
16]. Therefore, we speculated that the addition of nitrogen would have an impact on the floral traits of perennial plants, leading to fluctuations in seed production and reproductive efficiency on an annual basis.
Based on the available empirical data, it can be deduced that there has been a significant increase in global atmospheric nitrogen inputs [
16]. In Europe and North America, the nitrogen (N) addition rate commonly observed ranges from 10 to 25 kg N ha
-1 year
-1 [
17]. However, it is important to acknowledge that in China, the yearly rate of nitrogen addition is approximately 50 kg N ha
-1 year
-1 [
18]. The critical load of nitrogen (N) required to elicit a response from alpine meadow communities has been determined to be 10 kg N ha
−1 yr
−1 [
19]. The wet deposition of nitrogen has been observed to vary across different regions, with 6.69 kg N ha
−1 yr
−1 on the western Tibetan Plateau and 7.55 kg N ha
−1 yr
−1 on the eastern Tibetan Plateau [
20]. The examination of the responses exhibited by high-altitude ecosystems to elevated nitrogen deposition is highly appropriate for investigation on the Tibetan Plateau.
Herein, we proposed the hypothesis that the addition of nitrogen would have an impact on the annual fluctuations in the seed production of perennial plants, consequently leading to an improvement in their overall reproductive efficacy. In order to evaluate the veracity of this hypothesis, a multiyear field experiment was conducted utilizing the
S. nigrescens in Tibetan meadow. The species under investigation exhibits a perennial life cycle and is dependent on honey bees for the purpose of pollination [
21]. The objective of this study is to investigate the impacts of nitrogen supplementation on various aspects of plant traits, including the aboveground biomass, patterns of resource allocation, production of flowers and nectar, pollinator visitation, and seed production over multiple years. The expectation was that these factors would collectively have an impact on the interannual variation in seed production. The results of our study hold promise for advancing our understanding of the effects of nitrogen addition on the reproductive efficiency of perennial herbaceous plants, as well as the underlying mechanisms that regulate biodiversity in the context of global environmental changes.
3. Results
The vegetative and floral traits of
S. nigrescens exhibited interannual variability, which was found to be influenced by the amount of nitrogen in the soil (
Figures S1 and S2, and
Tables S1 and S2). The N4 and N8 treatments exhibited the greatest aboveground biomass, stem mass and capitulum mass in the third and fourth years, whereas no significant variation in above-ground biomass was observed across the years for the N0 treatment (
Figure S1). The number of capitula per plant and the number of flowers per capitulum reached their highest levels during the second and third years for the N4 treatment and in the third and fourth years for the N8 treatment. The highest recorded nectar volume per capitulum was observed in the first three years for the N0 treatment, while for the N8 treatment, it was observed in the third and fourth years. On the other hand, there was no statistically significant variation in nectar volume per capitulum for the N4 treatment across different years (
Figure S2). The addition of nitrogen did not result in any year-to-year variation in nectar concentration (
Figure S2).
The introduction of nitrogen had an impact on both the rate of pollinator visitation and seed set (
Figure S3,
Table S3). Peak pollinator visitation and seed set were observed during the initial two years for the N0 treatment and in the third and fourth years for the N8 treatment. Conversely, no discernible differences in these variables were found among the years of the N4 treatment (
Figure S1).
The reproductive efficiency and seed production per capitulum or plant were found to be influenced by the nitrogen supply in the soil (
Figure 1a,b;
Figure S4;
Table S3). The N0 treatment exhibited the highest reproductive efficiency and the greatest number of seeds per capitulum during the initial two years. Similarly, the N4 treatment demonstrated these characteristics in the second and third years, while the N8 treatment displayed them in the third and fourth years.
The introduction of nitrogen had varying effects on the correlations between different traits (
Figure 2). The study revealed a positive correlation between seed production per plant and both flower production per capitulum and capitulum production per plant (
Figure 2). There exists a positive correlation between the number of seeds per capitulum, the frequency of pollinator visits, and the quantity of nectar produced by each capitulum. The study revealed a positive correlation between above-ground biomass and various factors, including reproductive efficiency, seed set, pollinator visitation frequency, and the number of capitula per plant (
Figure 2).
The plant's overall seed production was observed to be significantly impacted by both the seed number per capitulum and the total number of capitula (
Figure 1c). Both the level of nitrogen addition and the experimental year had an impact on the quantity of seeds that a plant produced (
Figure 1c).
The results of model selection indicated a positive relationship between pollinator visitation rates and the number of flowers and capitula on a plant (
Figure 3).
4. Discussion
The findings indicated that increasing the nitrogen content in the soil has the potential to alter the natural fluctuations in seed production and reproductive efficiency that occur over different years of S. nigrescens. Nitrogen has been found to alter the interannual variations in the number of flowers per capitulum as well as the number of capitula per plant, consequently affecting the annual frequency of seed production. However, the introduction of nitrogen had an impact on the fluctuation of flower rewards from year to year. This included changes in nectar production per capitulum and the number of flowers per plant. Additionally, the patterns of pollinator visitation rate and seed set were also altered throughout the duration of the experiment. A positive correlation has been observed between the quantity of seeds produced per plant and several factors, such as the number of flowers per capitulum, the number of capitula per plant, the volume of nectar per capitulum, and the rate of visitation by pollinators. A significant positive correlation was identified between aboveground biomass and several factors, encompassing the allocation of biomass to stem, the number of flowers per capitulum, the number of capitula per plant, the volume of nectar per capitulum, and the seed production per plant. This implies that the incorporation of element N significantly influences the maintenance of aboveground biomass and flower traits, resulting in alterations in the frequency of pollinator visits and the interannual variability in seed production. The findings of this research have the potential to enhance our comprehension of the impacts of nitrogen supply on the reproductive efficacy of perennial herbs, as well as the underlying mechanisms that influence biodiversity in alpine meadows amidst global environmental changes.
The presence of nitrogen significantly influences plant biomass through the alteration of carbohydrate reserves and the allocation of carbon resources [
11,
12]. Plants that are grown in habitats that have an adequate nitrogen supply often exhibit a greater allocation of biomass towards the stem [
30,
31]. This allocation is necessary to provide mechanical support for the increased aboveground biomass of
S. nigrescens. In addition, the introduction of nitrogen addition resulted in an increase in community height (30~35 cm). This increase in height can be attributed to the promotion of stem growth, which enables
S. nigrescens to better compete for light resources [
32,
33]. Moreover, the highest aboveground biomass was observed during the third and fourth years of our study (
Figure S1). We postulated that a higher percentage of underground resources experienced a transformation resulting in the production of a greater amount of aboveground biomass over the course of the latter two years [
31].
Furthermore, plants frequently allocate a greater number of resources towards the development of their reproductive organs, including the production of a larger quantity of flowers and the provision of increased flower rewards [
9,
10]. This relationship is exemplified by a positive association between the aboveground biomass and several floral traits, such as the number of capitula per plant, the number of flowers per capitulum, and the amount of nectar per capitulum (
Figure 2). Both the N4 and N8 treatments exhibited an increase in aboveground biomass, as well as an increase in the flower mass fraction (
Figure S2). This implies that an augmentation in aboveground biomass has the potential to enhance the quantity of flowers and nectar [
13,
35]. Furthermore, our study revealed a positive correlation between nectar and flower production, and aboveground biomass fluctuations over consecutive years. The flower or capitulum number and nectar production of the plant and capitulum exhibit interannual variation, which is influenced by changes in aboveground biomass over the course of the experimental years. This finding suggests that the addition of nitrogen not only alters the year-to-year fluctuations in aboveground biomass [
31], but also affects the allocation of resources towards flower and nectar production, consequently leading to annual changes in flower and nectar abundance.
In a research investigation examining the impacts of nutrient supply on nectar traits, the addition of nitrogen did not result in an increase in nectar secretion for
Trifolium pratense, however, it did lead to an increase in the rate of nectar secretion for
Antirrhinum majus [
36]. Nevertheless, the augmentation in nectar secretion of
A. majus and
Ipomopsis aggregata was solely observed under conditions of minimal nitrogen supplementation (10 kg N ha
−1 year
−1). At elevated levels of nitrogen addition (200 kg N ha
−1 year
−1), there was a notable reduction in nectar secretion for both species [
13,
36]. This suggests that the influence of nitrogen supplementation on nectar production is dependent on both the dosage of nitrogen and the particular plant species. The nectar secretion of
S. nigrescens was found to be enhanced by both the N4 and N8 treatments. The extent to which the dose of nitrogen reduces nectar production remains unknown. Indeed, numerous studies have demonstrated that the introduction of nitrogen has a positive impact on the overall concentration of amino acids [
37,
38,
39]. The presence of nitrogen addition has been frequently observed to result in an increase in the levels of asparagine and glutamine, among the various individual amino acids [
15,
37,
39]. The disparity in nectar concentration (e.g., sugar content) was not identified in
S. nigrescens. The concentration of nectar is subject to influence by microclimate factors, particularly relative humidity [
40]. The available evidence indicates that the concentration of nectar is often influenced by its water content. The evaporation rate is influenced by the humidity gradient, which in turn affects the exchange of water between nectar and air [
41], ultimately impacting the concentration of nectar. The study site where three treatments were conducted exhibited similar levels of humidity. The plants exhibited uniform growth under equivalent relative humidity conditions, leading to nectar concentrations that were indistinguishable.
Bees derive their energy from the consumption of nectar and pollen. The provision of protein and other essential nutrients is imperative for the optimal growth and development of larvae. Plants that exhibit a high production of nectar in substantial quantities have the potential to attract a greater number of visits from pollinators, as well as prolong the duration of the visits [
21,
42,
43]. Within the designated study areas, it has been observed that honey bees play a predominant role as the principal pollinators of
S. nigrescens. There was a correlation between honey bee visitation and nectar production [
44]. Plants that thrive in N4 and N8 habitats, characterized by the presence of nectar-rich flowers, exhibit a higher frequency of pollinator visits. Conversely, plants inhabiting the N0 habitat, which is characterized by the absence of nectar-poor flowers, tend to attract fewer pollinators (
Figures S2 and S3). A positive correlation has been observed between the overall sugar concentration in nectars and specific pollinators [
45]. As an illustration, honey bees exhibit a preference for nectar characterized by a diminished sugar concentration [
46], while bumble bees display a preference for nectar characterized by an elevated sugar concentration [
47]. The sugar concentration of
S. nigrescens exhibited a range of 37% to 50%, aligning with the nectar concentration preferences observed in honey bees. The frequency of flower visits is impacted by the level of flower production. The findings from our model selection analysis indicated that both the quantity of nectar produced per plant and the number of capitula per plant are significant factors in determining the visitation of pollinators. It is imperative to acknowledge that floral scents have a significant impact on the attraction of pollinators [
48]. Nevertheless, the impact of nitrogen supplementation on the olfactory traits of flowers and the preferences exhibited by pollinators remains uncertain. The visitation rate is contingent upon the combined factors of species richness and abundance of pollinators [
49]. The honey bee population size exhibited no discernible variation among the study sites, as a result of the substantial release of honey bees by beekeepers in the designated study areas [
21,
22].
The introduction of nitrogen has the potential to modify the inherent variations in seed production, and reproductive efficiency observed across different years in
S. nigrescens. The presence of nitrogen has been observed to have an impact on the interannual variations in both the quantity of flowers per capitulum and the number of capitula per plant (
Figure 2). The seed yield per plant is influenced by two factors: the number of capitula per plant and the number of seeds per capitulum (
Figure 1).
S. nigrescens exhibits self-incompatibility, with honey bees serving as the primary pollinators for the purpose of fertilization [
21]. This deduction is supported by the demonstrated the positive correlation between seed set and pollinator visitation. The introduction of nitrogen resulted in a notable augmentation in the quantity of flowers and capitula, as well as an increase in nectar production and pollinator visitation [
13,
14,
16], consequently leading to an enhanced seed output. Moreover, there exists a correlation between aboveground biomass and the annual variation in seed production. A positive correlation has been identified between above-ground biomass and various reproductive traits, including seed quantity per plant, flower quantity per capitulum, and capitulum quantity per plant. This implies that the addition of nitrogen has the potential to modify the year-to-year fluctuations in aboveground biomass, resulting in changes in the number of flowers per capitulum, the number of capitula per plant, nectar production, and pollinator visitation across various years. Consequently, this can also impact the interannual seed output and reproductive efficiency.
Interannual variations in seed yield exert a significant impact on the sustainability of perennial plant populations [
1,
31]. In light of an uncertain environment, plants undertake a thorough evaluation of the benefits and costs linked to various reproductive strategies. Under optimal circumstances, plants exhibit a tendency to produce a larger number of seeds, thereby providing benefits for the replenishment of populations. Plants demonstrate a reduction in resource allocation towards reproduction when confronted with adverse circumstances [
50], opting to prioritize resource allocation towards survival. Our study suggests that over the course of the first two years of nitrogen supplementation, plants exhibited a preference for allocating resources towards vegetative tissue [
51]. This allocation strategy ultimately resulted in a significant improvement in seed yields during the subsequent third and fourth years for N4 and N8 plants.
The presence of soil nutrients has the potential to influence reproductive processes [
9]. For example, the presence of mycorrhizal infection in soil with high phosphorus content has the potential to enhance seed production [
52]. The present study did not observe the variation in soil nitrogen (N) and the other element levels among the different nitrogen supply treatments. It was found that soil nutrients exerted a notable impact on the secretion and concentration of nectar in flowers. Consequently, these factors influenced the rates of visitation by pollinators and the production of seeds [
13]. Further investigation would be required to conduct a comprehensive examination.
In general, augmenting the nitrogen composition in the soil possesses the capacity to modify the inherent variations in seed yield and reproductive efficacy observed across various years of S. nigrescens. However, the potential effects of nitrogen addition on the processes of seed germination and seeding establishment have yet to be investigated. The impact on plant abundance and community composition remains uncertain. The results of this study possess the capacity to augment our understanding of the effects of nitrogen supplementation on the reproductive efficiency of perennial herbaceous plants, as well as the fundamental mechanisms that impact biodiversity in alpine meadows in the face of global environmental changes.