4 | Discussion
Many studies have explored the response of flowering phenology to global
change. Three aspects of our study, however, distinguish it from these
previous studies. First, while a few studies of the effects of global
changes on phenology have considered one factors, our study examines the
effects of N addition, plant diversity loss, and their interactions on
plant flowering phenology in a single experiment. Second, our study
explores the effects of N addition and plant diversity loss on multiple
flowering phenology events. Third and most importantly, through three
different analysis methods, we confirmed that N addition and plant
diversity loss altered flowering phenology through its effects on
functional traits rather than abiotic factors.
4.1 | The effects of N addition and plant diversity
loss on flowering
phenology
The flowering phenology is one of the more important factors determining
the reproductive success of plants, because the timing of flowering
means when plants expose it reproductive organs to the changing biotic
and abiotic environments (Rathcke & Lacey, 1985; Kudo & Hirao, 2006).
In our study, N addition delayed the first flowering day (FFD) but
advanced the last flowering day (LFD), and then resulted in a shortened
flowering duration (FD), which consistent with those of previous
manipulative experiments in natural grasslands (Smith et al., 2012; Xi
et al., 2015). Nevertheless, the 1.92 d delay in FFD at the N addition
plots is shorter than that reported from the alpine grasslands in North
America (Smith et al., 2012); the 1.2 d advance in LFD and 3.2 d
shortened in FD is longer than that reported from the alpine grasslands
in China (Xi et al., 2015). Which suggesting that the effects of N
addition on flowering phenology varies among ecosystems and phenological
stages.
Different from the effects of N addition, plant diversity loss advanced
the FFD but delayed LFD, which resulted in an extension in FD. The 0.31
d advance per species lost in FFD is similar to the results (ranging
from an advancement of 1.8 d per species lost to a delay of 0.7 d per
species lost, average is 0.6 d advance) that reported from the
serpentine grasslands in North America (Wolf et al., 2017), suggesting
that the effects of plant diversity loss on FFD varies among species.
Moreover, we noticed that the amplitude of changes (0.64 d) in LFD is
more than that in FFD (0.31 d) after per species lost, which revealing
that LFD may be more sensitive to plant diversity loss.
4.2 | The effects of N addition and plant diversity
loss on functional
traits
We found evidence to support our second hypothesis that increased N
inputs promote light acquisition traits. Specifically, leaf mass and
area of M. sativa was promoted after N addition. These results were
inconsistent with that reported from the alpine grasslands in China (Liu
et al., 2017; Zhang et al., 2019), which may be induced by the
differences in species. M. sativa, a legumes, can fix N by means of
symbionts to relieve their N limitation (Bordeleau & Prévost, 1994),
and thus the nutrient acquisition traits of M. sativa had no changes
under N addition. Meanwhile, the relief of N limitation accompany with
the increasing demand of light (Hautier et al., 2009). Hence, the
changes in light demand of M. sativa under N addition could lead to an
increase in leaf mass and area.
Our findings for plant diversity loss also support our second hypothesis
that both light (specific leaf area) and nutrient (leaf nitrogen content
and relative abundance) acquisition traits was promoted under plant
diversity loss. These results were inconsistent with the effects of
plant diversity loss on grass species that reported from the grasslands
in Germany (Gubsch et al., 2011), which may be caused by the differences
in species. Closed canopies are characterized by pronounced gradients in
spectral light quality and quantity (Jones, 1992). In the present study,
plant diversity loss impacts on morphological traits associated with
light acquisition were mainly attributable to the changes in relative
height of M. sativa. Relative height decreased with plant diversity
loss, indicating higher efforts for light acquisition should be
allocated by plants (Spehn et al., 2005; Lorentzen et al., 2008). Hence,
M. sativa exhibited high specific leaf area in adjustment to increasing
competition for light. Moreover, nitrogen is not the limitation resource
of M. sativa. Higher light acquisition traits are accompanied with
higher nutrient acquisition traits (Hautier et al., 2009; Zhang et al.,
2019), which resulted in an increase in leaf nitrogen content and
relative abundance under plant diversity loss.
4.3 | Functional traits rather than abiotic factors
determine flowering
phenology
Our findings not support for our third hypothesis that functional traits
and abiotic factors co-diver the response of flowering phenology to N
addition and plant diversity loss, the contributions of functional
traits for changes in flowering phenology was far more larger than
abiotic factors (Fig. 5, 6, and 7). Specifically, the advance in FFD
under biodiversity loss was mainly induced by the decrease in available
soil N, which was consistent with the result from Wolf et al. (2017) in
American serpentine grassland. Lower available soil N advanced the plant
switches from the growth to reproduction stage (Wang & Tang 2019), and
then indirectly caused the advancement in FFD at the monocultures.
However, the response of LFD and FD to biodiversity loss and N inputs
was mainly driven by leaf nitrogen content and relative abundance. It
has been proved that plant morphological and physiological traits could
elucidate the changes in plant phenology (Jia et al., 2011; König et
al., 2018; Pérez-Ramos et al., 2019) and the alteration of living
strategies of plants (Wilson & MacArthur, 1967; He et al., 2008;
Pérez-Ramos et al., 2019). At the monocultures, higher efforts for
nutrients (leaf nitrogen content and relative abundance) acquisition
means increase in intraspecific competition for resource among
individuals of M. sativa (Lorentzen et al., 2008; Liu et al., 2017), and
K-strategy with higher survive rate was chosen by M. sativa at the
monocultures, which resulted in delaying LFD, extending FD, and
decreasing FN (Fig. 2). As species richness increase, the intraspecific
competition for resource decrease, but the interspecific competition
among different species increase (Hector et al., 1999; Tilman et al.,
2001). However, because the predominant competition advantage of M.
sativa (high relative abundance and plant height, Fig. 4) at the
mixed-cultures, r-strategy was chosen by M. sativa with more flower
numbers and lower survival rate (Guiz et al., 2018), which resulted in
advancing LFD, shortening FD, and increasing FN (Fig. 5b, c, and d).
Therefore, the gradually shifts from r-strategy to K-strategy of M.
sativa led to the changes in flowering phenology under plant diversity
loss.