1 | Introduction
Human activities such as agricultural production and fossil fuel
combustion add ~200 Tg/year of reactive nitrogen (N) to
global ecosystems, which is approximately equal to that provided by
natural N fixation (Galloway et al., 2004; Schlesinger, 2009; Fowler et
al., 2013). Apart from N deposition increasing, biodiversity loss is
another global change issues that human being confront, which is mainly
induced by land use change, climate change, and N deposition increase
(Sala et al., 2000; Gossner et al., 2016; Harpole et al., 2016). Recent
study showed that the world’s seed-bearing plants have been disappearing
at a rate of nearly 3 species a year since 1900, which is up to 500
times higher than would be expected as a result of natural forces alone
(Ledford, 2019). It has been proved that the plant life cycles and
ecosystem functions is significantly changed under N deposition increase
and plant diversity loss (Hautier et al., 2014; Grace et al., 2016; Wolf
et al., 2017; Pennekamp et al., 2018), yet how N addition, biodiversity
loss, and its interactions influence on plant phenology remains
uncertain.
Flowering phenology, including flowering date and duration, is important
developmental stages in plant phenology as well as a sensitive indicator
of global changes (Hovenden et al., 2008; Hulme 2011; Wang et al., 2014;
Suonan et al., 2017; Piao et al., 2019). Many factors control the
flowering phenology in grassland ecosystems, N deposition increase and
biodiversity loss are two of the most important (Sanz et al., 2011;
Smith et al., 2012; Xia & Wan, 2013; Xi et al., 2015; Wolf et al.,
2017; Wang & Tang, 2019a). While numerous studies reveal that N
addition delayed the flowering phenology of plant (Smith et al., 2012;
Xia & Wan, 2013; Wang & Tang, 2019a), and plant diversity loss
advanced flowering events (Wolf et al., 2017). Specifically, increasing
N inputs lead to an increase in available soil N (Smith et al., 2012),
and then could delay flowering phenology indirectly through extending
the length of vegetative growth (Wang & Tang, 2019a). While plant
diversity loss could influence flowering phenology indirectly, via
effects on abiotic processes (soil moisture and temperature) and
resource availability (available soil N), or directly, via biotic
interactions (plant density) (Wolf et al., 2017; Du et al., 2019). The
projected N addition and plant diversity loss could change abiotic and
biotic processes in the community and thus impact plant flowering
phenology.
Functional traits of a plant are measurable biotic properties related to
adaptation to environment (Enquist et al., 2015; He et al., 2018;
Gustafsson & Norkko, 2019). Compared with abiotic factors, the
alteration in biotic factors (e.g. functional traits) may be the more
directly and better indicator of ecosystem function changes under N
addition and plant diversity loss (Dickson et al., 2014; Cadotte, 2017).
Given that soil N availability often alters the switches from the growth
to reproduction stage (Cleland et al., 2006), and thus increasing N
could delay flowering phenology (Wang & Tang, 2019a); available soil N
increase under plant diversity loss, and then plant species flowered
earlier, with peak flowering date advancing an average of 0.6 days per
species lost (Wolf et al., 2017). However, previous studies showed that
the indirectly effects of abiotic factors on phenology through altering
plant morphological and physiological traits (König et al., 2018;
Pérez-Ramos et al., 2019). Plant light and nutrient acquisition traits
could elucidate linkages between plant phenology and environmental
changes (Jia et al., 2011; Dorji et al., 2013; König et al., 2018;
Pérez-Ramos et al., 2019). For instance, traits associated with resource
acquisition, such as rooting depth and life history, mediate plant
reproductive phenology responses to changing climatic conditions in an
alpine grassland (Dorji et al., 2013); traits related to competition and
growth rate, like plant height, specific leaf area and leaf dry matter
content, had substantial explanatory power in the effects of climate
changes on the first flowering day at the global scale (König et al.,
2018; Pérez-Ramos et al., 2019). Therefore, our knowledge regarding the
linkages between changes in flowering phenology and functional traits is
crucial for better understanding the regulating mechanisms of N
enrichment and plant diversity loss on plant phenology and predicting
grassland community dynamics under increasing N deposition and
biodiversity loss scenarios.
Here, we report on a common garden experiment investigating the
influence of N deposition increase and plant diversity loss on three
flowering phenology of Medicago sativa in an assemblage grasslands
(Supplementary Fig. 1). Specifically, we explored the effects of N
addition and multiple plant diversity levels on the first flowering day,
the last flowering day, flowering duration, flower numbers,
environmental factors, and a series of plant functional traits. We used
partial correlation, variation partitioning, and structural equation
modelling analysis to quantify the direct and indirect effects of N
addition and plant diversity loss on flowering phenology and flower
numbers. We hypothesized that (a) increased N inputs and plant diversity
would delay flowering phenology; (b) increased N inputs promote light
acquisition traits, and decreased plant diversity promote light and
nutrient acquisition traits; and (c) changes in abiotic factors and
functional traits would together drive the response of plant flowering
phenology to N addition and plant diversity loss.