Phenological responses to nitrogen and water addition are linked to plant growth patterns in a desert herbaceous community

Abstract Increases in nitrogen (N) deposition and variation in precipitation have been occurring in temperate deserts; however, little information is available regarding plant phenological responses to environmental cues and their relationships with plant growth pattern in desert ecosystems. In this study, plant phenology and growth of six annuals in response to N and water addition were monitored throughout two consecutive growing seasons in 2011 and 2012 in a temperate desert in northwestern China. The effects of N and water addition on reproductive phenology differed among plant species. N and water addition consistently advanced the flowering onset time and fruiting time of four spring ephemerals; however, their effects on two spring‐summer annuals were inconsistent, with advances being noted in one species and delays in another. N and water addition alone increased plant height, relative growth rate, leaf number, flower number, and individual biomass, while their combinative effects on plant growth and reproductive phenology were dependent on species. Multiple regression analysis showed that flowering onset time was negatively correlated with relative growth rate of two species, and negatively correlated with maximum plant height of the other four species. Our study demonstrates that phenological responses to increasing precipitation and N deposition varied in annuals with different life histories, whereby the effects of climate change on plant growth rate were related to reproductive phenology. Desert annuals that were able to accelerate growth rate under increasing soil resource availability tended to advance their flowering onset time to escape drought later in the growing season. This study promotes our understanding of the responses of temperate desert annuals to increasing precipitation and N deposition in this desert.


| INTRODUC TI ON
Phenology is the onset time and duration of biological events, and it determines species fitness and coexistence in plant communities (Forrest & Miller-Rushing, 2010). Plant phenology is sensitive to variation in the ambient environment, and phenological shift as a result of species acclimation to environmental conditions (Meineri, Skarpaas, Spindelbock, Bargmann, & Vandvik, 2014;Parmesan & Yohe, 2003;Sparks & Carey, 1995) can trigger a series of changes in plant reproduction, community composition, and even ecosystem functions (Cleland, Chuine, & Menzel, 2007;Huang & Li, 2015;Schwartz, 2003). Plant phenological shift can affect competitive interactions among plants and the stability of trophic levels, thus consequently influencing ecosystem processes, including nutrient fluxes (Fogelstroem et al., 2017;Leverett, 2017).
Increasing atmospheric N deposition has been occurring on a global scale due to the combustion of fossil fuels and application of N fertilizer in agricultural ecosystems (Galloway et al., 2008). N deposition alters plant carbon assimilation, distribution, and growing season (Liu, Miao et al., 2017;Llorens & Penuelas, 2005), but the effects are species specific (Liu, Miao et al., 2017, Liu, Monaco et al., 2017Peñuelas et al., 2004;Sherry, Zhou, & Gu, 2007;Xia & Wan, 2013;Zhang, Niu, Liu, Jia, & Du, 2014). For example, N addition advanced the flowering onset time and fruiting time of two spring-summer annuals in the Chihuahuan Desert (Whitford & Gutierrez, 1989), as well as some forbs and a legume in a North American grassland, but had no impact on other plants (Cleland et al., 2006;Sherry et al., 2007).
Simultaneous variation in N and water availability can have synergistic effects on plant growth and phenology as decreased water availability might hamper N addition effects in arid and semiarid regions (Crimmins, Crimmins, & Bertelsen, 2010;Nord, Shea, & Lynch, 2011;Peñuelas et al., 2004;Sherry et al., 2007). Although different climate changes and environmental factors altered by anthropogenic activity, including precipitation and N, usually co-occur, few studies have investigated the interactions of two or more factors (Liu, Miao et al., 2017, Liu, Monaco et al., 2017Nord et al., 2011;Schuster & Dukes, 2017;Sherry et al., 2007). Therefore, manipulative field experiments are needed to investigate individual and combined effects of precipitation and N on plant growth and reproductive phenology in temperate deserts.
Plant responses to environmental changes can often be generalized by plant life-history strategy. For example, annual grasses show earlier flowering onset time than forbs (Cleland et al., 2006), and early-flowering species showed advanced flowering in response to warming, while late-flowering species exhibited delayed flowering (Sherry et al., 2007). In general, plant species' strategies in dry lands can be divided into drought avoidance and drought resistance, based on their water use. However, it is unclear how plant phenology responds to water or N availability in terms of life-history strategy.
There are three typical plant growth patterns in response to resource availability in desert ecosystems: Drought-escape ephemerals only use soil resources at the first nutrient and water pulse in early spring; however, such a one bet strategy carries a risk of utter reproductive failure if resources are only available for a very short period (Figure 1curve A). Drought-resistant spring-summer annuals can endure drought during their reproductive period, and benefit from the second resource pulse in a low competition community because of the mortality of spring ephemerals (Curve B). Finally, drought avoiding spring-summer annuals can survive drought periods through slow growth or aboveground dormancy and maintain reproduction in later resource pulses in autumn (Curve C). Accordingly, species with these distinct life-history strategies may respond differently to water or N availability and thus modify the species coexistence in desert ecosystems (Rasmussen, Van Allen, & Rudolf, 2014;Zhang, Hu, & Zhang, 2016).
Given the importance of resource pulses for desert ecosystems, climate changes could have a profound effect on plant communities and ecosystem function. We investigated this using a manipulative experiment in a desert herbaceous community in northwestern China. Precipitation in northwestern China has been increasing over the past 50 years, and this trend is predicted to persist in the near future (Cholaw, Cubasch, & Lin, 2003;Ni & Zhang, 2000). Moreover, airborne N deposition increased from 1.3 g N m −2 year −1 in 1980 to 3.5 g N m −2 year −1 in 2012, and is expected to continue increasing primarily because of N fertilizer application to the encroaching farmland Zhang, Zheng, & Liu, 2008 (Su, Li, Cheng, Tan, & Jia, 2007;Su, Wu, Zhou, Liu, & Zhang, 2013). The soil of inter-dune land is sandy loam and derived from alluvial deposits. The sandy dune soil is generally eolian.
The annuals can be grouped into spring ephemerals and springsummer annuals in terms of their life-history strategy, with a coverage reaching 40% at peak plant growth ( Figure 2). Spring ephemerals germinate immediately after snowmelt in mid-late March, mature in mid-May, and die off in late May-early June. Spring-summer annuals usually exhibit low growth rates as accompanying species during the main growth period of spring ephemerals and show fast growth rates after the mortality of spring ephemerals.

| Experimental set-up
This study combined simulated N deposition and increased precipi-  pan" installed beside each plot (15% of the plot area). The pan was erected with galvanized iron sheets at a slight angle of 2°, so that the collected rain could run into a plastic bucket buried in the soil.
Collected rain was evenly sprayed onto the corresponding plot as soon as possible to prevent evaporation losses, usually during late afternoon or early morning after rainfall.

| Environmental variable measurement
We measured air temperature, precipitation, soil moisture, and inorganic N content. Air temperature and precipitation were monitored hourly by an automatic meteorological station (Campbell Science Equipment, Logan, UT) at our study site. Soil moisture was monitored biweekly using time domain reflectometry in each plot at 0-20 cm depth (Diviner-2000, Sentek Pty Ltd., Balmain, Australia).
Soil water content reaches saturation immediately after snowmelt (volumetric water content 12%), and gradually decreases throughout May and June. When soil water content was below 5% and lasted >1 week, we defined this period of low soil moisture as a drought period ( Figure S1). For soil inorganic N content, soil cores (5 cm in diameter and 5 cm in depth) were collected monthly in April-September in each plot and composited to make one per plot. Samples were placed in plastic bags, transferred to the lab and stored at 4°C for further analysis. Soil dissolved inorganic N (DIN, including nitrate-N (NO 3 − -N) and ammonium-N (NH 4 + -N)) was extracted from 10-g fresh soil with 50 ml 2 mol/L KCl. After shaking for 1 hr, DIN was filtered and the filtrate was measured using a continuous-flow ion auto-analyzer (AA3, Bran Luebbe, Germany).

| Phenology and plant growth observations
The six species used for plant phenological observation accounted for more than 84% of the total coverage and 75.7% of the herbaceous biomass at our study site. The six species can be grouped into two    following the modified method described by Price and Waser (1998), Dunne, Harte, and Taylor (2003), and Sherry et al. (2007).
Plant height, leaf number, and flower number were also measured. In addition, individual biomass was calculated using the measured height and the allometric growth equation for each species from a previous study (Huang, Su, Zhu, & Li, 2016). Finally, plant density was obtained by recording the number of plants in a 5 m 2 area in each plot at peak plant growth.

| Statistical analysis
The Richards growth equation was used with the contractionexpansion algorithm to describe the phenological data of each species within the plots (Richards, 1959): where Y is the scored phenological stage, K is the maximum growth Relative growth rate was calculated using the following equation: where t 1 is the day at which the species first appeared) and t 2 is the day at first flowering; and H 1 and H 2 are the height (in cm) of the plant at t 1 and t 2 , respectively.
Initially, five-way ANOVA was used to examine the effects of .

| Nitrogen and water addition effects on soil inorganic N content and soil moisture
N addition significantly increased soil inorganic N and nitrate-N, while exerting no impacts on soil ammonium-N (Table 1). N addition had no impacts on soil moisture (Table 1). Water addition slightly increased soil nitrate-N and ammonium-N (Table 1), and significantly increased soil moisture during the spring ephemeral growing period from April to June in both 2011 and 2012 (Table 1). Water addition significantly increased soil moisture at 0-10 cm by an average of 1.9% in 2011 and 2.9% in 2012 ( Figure   S1). Combined addition of N and water significantly increased soil inorganic N, but the accompanying increase in soil moisture was not significant (Table 1).

| Nitrogen and water addition effects on plant phenology
N and water addition alone and in combination affected flowering onset time, but the effect varied by species (Table 2). Across  (Table 2); however, it significantly advanced fruiting time of M. scorpioides (4 days), and delayed that of S. brachiata (11.6 days; Figure 3). N and water addition together had significant interactive effects on fruiting onset time of C. arenarius across the two years (Table 2).
N addition did not alter seed maturation time, when both growing seasons were analyzed (Table 2) Water addition significantly affected seed maturation time and interacted with species (Table 2), with significant impacts on C. arenarius (Figure 3). N and water addition had no interactive effects on seed maturation time of any species (Table 2).
Both N and water addition significantly altered reproductive duration (Table 2) Table 2). The reproductive duration was significantly shorter in 2012 compared with that in 2011 for all species except S. brachiata, which showed no difference between the two years ( Figure 4).

| Nitrogen and water addition effects on plant growth
N addition significantly promoted maximum plant height and leaf number (  (Tables S1 and   S2). Water addition promoted the maximum plant height of all species in two years (Table 2). Water addition also promoted plant relative growth rate and leaf number, but this varied by species and year (

| Relationship between plant phenology and growth
Flowering onset time was negatively related to plant maximum height in all species except A. linifolium and L. filifolium (Table 3).

Flowering onset time of spring ephemerals and fruiting time of
A. linifolium, E. oxyrrhynchum, and C. arenarius were negatively related to plant relative growth rate (Table 3). Flowering onset time of spring-summer annuals was negatively related with maximum height (Table 3). Flowering onset time and fruiting time of spring-summer annuals were both negatively correlated with leaf and flower numbers (Table 3). There was no correlation between seed maturation time and plant growth traits except the correlation between seed maturation time and relative growth rate for E. oxyrrhynchum, M. scorpioides, and C. arenarius (Table 3).
Reproductive duration was positively correlated with relative growth rate in A. linifolium and M. scorpioides and with plant maximum height in C. arenarius (Table 3) (Table 3).

Plant growth traits
Stepwise Stepwise regression analysis was further used to establish the relationships between phenology and growth traits. Asterisks indicate significant correlation between phenophases and growth traits at p < .05. / indicates no measurement was conducted. 2013). This may explain the high N absorption and the sensitive phenological responses (Phillips et al., 2013;Pregitzer, Burton, Zak, & Talhelm, 2008). The different phenological responses among species to N addition could have important ecological implications. For instance, the dramatic change in reproductive phenology can induce ecological asynchronies in plant community, thereafter affecting their pollinators or herbivores (Hegland, Nielsen, Lázaro, Bjerknes, & Totland, 2009;Inouye, 2008). Moreover, the differential responses among species to varying environmental cues can reflect and influence community resistance to disturbance (Stevens & Carson, 2001).

| Flowering and fruiting responses to water addition
Our study showed distinct responses in flowering onset time to a

| Flowering and fruiting responses to water plus nitrogen addition
Few studies have addressed the combined effects of N and water addition on reproductive phenology of desert annuals (Cleland et al., 2007;Gutierrez & Whitford, 1987;Liu, Monaco et al., 2017;Mauritz, Cleland, Merkley, & Lipson, 2014;Sharifi et al., 1988). Our study shows that the combination of N and water addition only advanced flowering onset time and fruiting time of C. arenarius. No interactive effects on spring ephemerals were noted for two reasons: First, water addition may have promoted soil N mineralization (Austin, Yahdjian, & Stark, 2004) or increased N leaching in the rhizosphere, as evidenced by the similar soil inorganic N content in water addition and control plots (Huang et al., 2016). Second, plant phenology is associated with the date of snowmelt (Philipp et al., 2016) because snowmelt in early spring usually equates to 40 mm rainfall, and has an overriding effects on seed germination of spring ephemerals in this desert (Fan, Tang, Wu, Ma, & Li, 2014). Further study is needed to test the interactive effects of snowmelt and N addition on the reproductive phenology of desert annuals.
Plant functional traits can be used to predict the community responses to climate changes. For example, N addition advanced flowering onset time of some forbs but delay that of grasses in North American grassland (Cleland et al., 2006). Similarly, the response magnitude of flowering onset time to the variation in precipitation varies among species (Cleland et al., 2006(Cleland et al., , 2007Lasky et al., 2016;Prieto, Peñuelas, & Ogaya, 2008;Schwartz, 2003;Xia & Wan, 2012  Plant relative growth rate can reflect plant survival strategies (Adler et al., 2014). Flowering is an indicator of switching from vegetative growth to reproductive growth, and plants usually prepare extensively for flowering via growth and mass storage (Schwartz, 2003). In our study, Flowering onset time was negatively correlated with the relative growth rate across six dominant species (Huxman, Barron-Gafford, & Gerst, 2008;Kimball, Angert, & Huxman, 2011;Sun & Frelich, 2011), indicating that spring ephemerals tend to flower earlier than spring-summer annuals. Given the phenological variation within a community, strategies adopted by spring-summer annuals under frequent drought may be the crucial intrinsic mechanism that explains phenological responses to combined addition of water and N in deserts (Campanella & Bertiller, 2008;Dorji et al., 2013;Meineri et al., 2014;Sakkir, Shah, Cheruth, & Kabshawi, 2015). We thus deduced that drought-escape species, like spring ephemerals, may accelerate plant growth and advance plant phenology under abundant soil moisture and N (Aronson et al., 1992;Fox, 1990;Kemp, 1983), while drought-resistance species, like C.

| CON CLUS ION
Our study shows that N and water addition modified a set of plant phenological events and growth traits, and the response direction and magnitude of reproductive phenology are closely related to plant growth traits. Both N and water addition advanced the flowering onset time and fruiting time of spring ephemerals by promoting plant growth rate, although N and water had inconsistent effects on spring-summer annuals. Additionally, N and water had no synergistic effects on plant phenology. The stimulatory effect of water addition on plant growth suggests a preliminary role of precipitation in shaping the composition and structure of a desert herbaceous community. More importantly, the advancement and expansion of reproductive phenology caused by increasing precipitation connected the gap of reproductive phenology at the community level during a dry year, which can buffer the negative effects of drought in the desert ecosystem. Thus, prolonged plant phenology and the promotion of plant growth and seed production in response to increasing precipitation will most likely benefit community composition and stability in the context of global climate change in temperate deserts.

ACK N OWLED G M ENTS
We greatly appreciate anonymous reviewers for their time and valuable comments on our paper, Dr. Sayer for careful edits on our paper, Drs. Yangui Su for data analysis, Zhong-dong Lan and Lei Chen for experimental instrument installation, Xin-jun Zheng, Jiang-bo Xie, and Jie Ma for field data collection. This study was sponsored by the National Natural Science Foundation of China (No. U1703332, 41671114, 31570455), Key Research Program of Frontier Sciences, CAS (QYZDJ-SSW-DQC014), and Youth Innovation Promotion Association, CAS (2016381).

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R CO NTR I B UTI O N S
GH collected data and wrote the paper, CHL review the paper, YL designed the research.