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Effects of nitrogen addition and mowing on reproductive phenology of three early-flowering forb species in a Tibetan alpine meadow
Ecological Engineering 99 (2017) 119–125
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Effects of nitrogen addition and mowing on reproductive phenology of three early-flowering forb species in a Tibetan alpine meadow Yinzhan Liu a,1 , Renhui Miao a,1 , Auqun Chen a , Yuan Miao a , Yanjie Liu b , Xinwei Wu c,∗ a b c
International Joint Research Laboratory for Global Change Ecology, College of Life Sciences, Henan University, Kaifeng, Henan 475004, China Department of Biology, University of Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany Department of Ecology, College of Life Sciences, Nanjing University, Nanjing, Jiangsu, 210093, China
a r t i c l e
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Article history: Received 22 June 2016 Received in revised form 15 September 2016 Accepted 13 November 2016 Keywords: Flowering time Fruiting time Hay mowing Nitrogen deposition Qinghai-Tibetan plateau Standing litter
a b s t r a c t Nitrogen (N) deposition and land use are likely to alter plant phenology, with subsequent effects on community structure and ecosystem function. Phenological responses of three early-flowering species, Anemone trullifolia var. linearis, Caltha scaposa, and Trollius farreri to N addition and hay mowing were investigated in a Tibetan alpine meadow over three years. N addition significantly delayed the first flowering time of A. trullifolia var. linearis (by 11.7 days) and C. scaposa (by 11.1 days), but did not affect that of T. farreri. Mowing prolonged the first flowering time and first fruiting time by 4.7 and 7.4 days across all three species. Significant interactions between mowing and N addition on reproductive phenology characteristics were detected. Mowing induced changes in the first and last flowering times were 6.5 and 4.3 days earlier in the N addition plots than those in the N non-addition plots. The changes in reproductive time were mainly attributed to the variations in standing litter in each treatment, i.e. reproductive phenology (timing of flowering and fruiting) was positively associated with litter accumulation. Our results indicate that N deposition and land use can affect plant phenology by changing the accumulation of standing litter in Tibetan alpine meadows. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Plant phenology is the seasonal timing of environmentalmediated phenomenon, and therefore sensitive to environmental changes (Cleland et al., 2006; Neil and Wu, 2006; Wolkovich et al., 2012; Guo et al., 2013; Fu et al., 2015; Ge et al., 2015). Responses of plant phenology to environmental changes can alter community structure (Encinas-Viso et al., 2012), inter-species interactions (Rafferty and Ives, 2011), and ecosystem carbon exchange (Xia et al., 2015). Plant phenology is thus widely regarded as an important ˜ indicator of global change (Menzel and Fabian 1999; Penuelas and Filella 2001; Chuine et al., 2004; Keenan, 2015).
Abbrevations: FFlT, First flowering time; LFlT, last flowering time; FFrT, first fruiting time; LFrT, last fruiting time; AT, Anemone trullifolia var. linearis; CS, Caltha scaposa; TF, Trollius farreri. ∗ Corresponding author at: Department of Eology, College of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing, Jiangsu, 210023, China. E-mail address: [email protected] (X. Wu). 1 These authors contribute equally to this paper. http://dx.doi.org/10.1016/j.ecoleng.2016.11.033 0925-8574/© 2016 Elsevier B.V. All rights reserved.
As an important aspect of global change, nitrogen (N) deposition is regionally becoming serious (Galloway et al., 2004; Liu et al., 2013). N deposition has been demonstrated to facilitate plant photosynthesis (Fleischer et al., 2013) and stimulate plant growth (Xia and Wan, 2008; Cao et al., 2011; Lü et al., 2011; Zhang et al., 2013), which may further alter plant reproductive phenology (Cleland et al., 2006; Smith et al., 2012; Xia and Wan, 2013; Xi et al., 2015). For example, Cleland et al. (2006) reported that N addition prolonged flowering times of forbs but delayed those of grasses in an American annual grassland. Smith et al. (2012) found contrary results in an alpine tundra. Xia and Wan (2013) showed that N addition in a temperate steppe did not affect flowering time at the community level. Therefore, the effects of N deposition on plant reproductive phenology remain controversial across plant functional groups and species in different ecosystems. Mowing for hay is an important human activity in grazing pastures, and can change microclimates (Wan et al., 2002) and associated plant traits such as plant height and specific leaf area (Kahmen and Poschlod, 2002; Diaz et al., 2007; Klimesova et al., 2008), which are closely related to plant reproductive phenology (Sun and Frelich, 2011; Wolkovich et al., 2012). However, there is little evidence for the effects of mowing on plant reproductive
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phenology. We know only several cases in which mowing has been shown to advance plant flowering time to avoiding to be cut off because of the changes of selective forces during evolution (Reisch and Poschlod, 2008), or delay plant reproductive phenology by increasing the time to regenerate lost tissue (Freeman et al., 2003) and reduce the growth rate (Trtikova, 2008). Therefore, the effects of mowing on plant reproductive phenology are also unclear. The eastern part of the Tibetan plateau is characterized by extensive alpine meadows. This area has experienced universal N deposition (0.53 kg N ha −2 yr −1 ) in the last few years and it is predicted to consistently increase in the future (Liu et al., 2013). Meanwhile, these meadows provide large amounts of high-quality forages, supporting millions of livestock (Xiang et al., 2009). Mowing for hay is the only way for Chinese herdsman to store forages in winter (Cao et al., 2009). Mowing removes the aboveground part of the plant, which may alleviate the N induced changes in plant growth and alter associated plant reproductive phenology. Thus, N deposition and mowing might interactively affect plant reproductive phenology in these meadows. However, to the best of our knowledge, no study has examined the interaction between N addition and mowing in this area. In the present study, we conducted a manipulative 3-year field experiment involving N addition (addition vs. non-addition) and mowing (mowing vs. non-mowing) in a Tibetan alpine meadow. We quantified plant reproductive phenology traits including the first and last flowering times, the first and last fruiting times, and community standing litter height as well as flowering height of three early spring flowering species. These data were used to examine the effects of both N deposition and mowing on plant reproductive phenology of early-flowering forb species to predict responses of plant phenology to global change.
2. Materials and methods 2.1. Study site The study was performed in Hongyuan County (32◦ 48 N, 102◦ 33 E) of Sichuan province in the eastern Qinghai-Tibetan Plateau. The altitude is about 3500 m a.s.l. The climate is characterized by a short and cool spring, summer, and autumn and a long winter. Mean annual temperature is 0.9 ◦ C, with maximum and minimum monthly means being 10.9 ◦ C and −10.3 ◦ C in July and January, respectively. Mean annual precipitation is 690 mm, almost 80% of which occurs between May and August. The soil is classified as Mat Cry-gelic Cambisol (Chinese Soil Taxonomy Research Group 1995) with high soil organic carbon (250 g kg−1 ) but low total nitrogen (8 g kg −1 ) and phosphorus (5 mg kg −1 ) content at a depth of 0–20 cm (Wu et al., 2011). The study area is dominated by Blysmus sinocompressus, Carex enervis ssp. Chuanxibeiensis, Deschampsia caespitosa, Anemone trullifolia var. linearis, Potentilla anserina, Haplosphaera himalayensis, Aster alpinus and Gentiana formosa. In the growing season (late May to late September), the peak value of total community coverage is over 90% and average plant height is > 30 cm in late-August (Wu et al., 2011). In particular, Anemone trullifolia var. linearis, Caltha scaposa and Trollius farreri were the only three early-flowering perennial forb species in the study area (Liu et al., 2011), and occupied 22–61%, 0.23–1.1%, and 0.76–1.85% of the total biomass. The first flowering time of the three species happened from early May to early June, which was in the first two months of the snow-free season at the study site. The first fruiting time of A. trullifolia var. linearis, and C. scaposa occurred in late May, and the first fruiting time of T. farreri occurred in early August. We here focused on the responses of reproductive phenology of these three species to N addition and mowing.
2.2. Experimental design In a fenced area of 1 ha, we conducted an orthogonal two factortwo level experiment in which we crossed N addition (addition vs. non-addition) with mowing (mowing vs. non-mowing), yielding a total of four treatments: (i) neither N addition nor mowing (N-, M); (ii) N addition only (N+, M-); (iii) mowing only (N-, M + ); (iv) both N addition and mowing (N+, M + ). Each of these four treatments was replicated five times (n = 20 in total), each replicate was a 1.5 × 0.75 m2 plot. N (in the form of urea; 5 g N m−2 yr−1 ) was added to the nitrogen addition treatments in early April from 2008 to 2010. We cut and removed aboveground plant parts (leaving 5 cm) in early October after all phenological events were complete in the mowing treatments in both 2008 and 2009. In addition, soil temperature (5 cm underground) was measured in three plots for mowing verse non-mowing treatments using thermometers (model DS1921G, Maxim Integrated Products, Sunnyvale, California, USA). Over a 6-month duration (from January 19th to July 23rd, 2010), the mean daily temperature (over 24 h of measurements made every 30 min) in the mowing plots was 0.84 ◦ C higher than in the non-mowing plots (Fig. A. 1 in Supplementary material). 2.3. Standing litter The average standing litter height was calculated following the method by Liu et al. (2012). The number of each plant species (species level abundance) was investigated in a subplot of 0.5 × 0.5 m2 in each plot on 26th to 28th of August in 2009. The standing litter height of each species (from three individuals) was measured in each plot from 21th to 25th of March 2010. The community standing litter height was calculated using the following equation: SLH =
n
Hi ×
1
Ai
n
1
Ai
Where Hi and Ai stood for the standing litter height and the abundance of the species i, and n was the number of species. 2.4. Phenological observations Three flowering stems of each species were randomly taped in each plot and measured on 21th to 25th of March 2010. The flowering height (FlH) of each species was calculated as the mean value of the flowering stems. The number of flower and fruit (if any) of the three species in each plot was investigated over a 5-day interval from 1st April (before the earliest flowering time) to 27 August (after the latest fruiting time) in 2010. The first and last flowering/fruiting proportions (y) were calculated using a quadratic equation, following the protocols of Sun and Frelich (2011), i.e., y = ax2 + bx + c, where x is the Julian date. We defined the time when 10% and 90% of flower were blooming as the first flowering times (FFlT) and the last flowering times (LFlT), respectively. We defined the time when 10% and 90% of fruit were borne as the first fruiting times (FFrT) and the last fruiting times (LFrT), respectively. 2.5. Data analysis Three-way ANOVAs were employed to determine the effects of species, N addition and mowing on reproductive properties including FFlT, LFlT, FFrT, LFrT, and FlH. Two-way ANOVAs were performed to determine the effects of N addition and mowing on the species level reproductive properties including FFlT, LFlT, FFrT, LFrT, FlH, and the community level standing litter height (i.e., SLH). Once a significant effect was detected, post hoc LSD tests were used to further elucidate treatment differences. Where parametric tests
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Table 1 Results of ANOVAs showing the effects of mowing (M), nitrogen addition (N), species and their interactions on the first flowering times (FFlT), the last flowering times (LFlT), the first fruiting times (FFrT), the last fruiting times (LFrT), and flowering height (FlH) at the community level and the species level, df and F values are provided. ˆ , P < 0.1; * , P < 0.05; ** , P < 0.01; *** , P < 0.001. df
FFlT
LFlT
FFrT
LFrT
FlH
M N species M*N M * species N * species M * N * species
1 1 2 1 2 2 2
57.9*** 1.6 68.9*** 9.0** 10.8*** 8.4*** 0.6
17.7*** 2.6 262.6*** 4.3* 1.6 5.1* 0.5
41.8*** 2.0 105.6*** 0.1 4.0* 0.7 1.9
16.8*** 8.8** 172.5*** 1.9 1.3 5.4** 0.5
107.2*** 11.2** 102.2*** 6.7* 6.6** 1.9 0.6
Anemone trullifolia var. linearis M N M*N
1 1 1
44.6*** 5.3* 1.3
20.2*** 8.5* 0.5
23.6* 0.5 0.1
8.6** 1.8 0.1
51.6*** 7.2** 0.3
Caltha scaposa M N M*N
1 1 1
36.2*** 6.5* 2.1
6.3* 3.5ˆ 1.7
33.6*** 3.2ˆ 3.2ˆ
5.6* 11.0** 1.4
21.6*** 5.8* 4.3ˆ
Trollius farreri M N M*N
1 1 1
0.4 6.6* 6.6*
1.9 4.3ˆ 3.0
1.9 0.0 0.5
7.5* 0.1 0.5
34.4*** 0.3 5.5*
Fig. 1. First flowering times (FFlT, a), last flowering times (LFlT, b), first fruiting times (FFrT, c), and last fruiting times (LFrT, d) of three plant species (AT, Anemone trullifolia var. linearis; CS, Caltha scaposa; TF, Trollius farreri) among the four treatments (N-, nitrogen non-addition; N+, nitrogen addition; M-, non-mowing; M+, mowing). Error bars are means ± SE.
were performed, Kolmogorov-Smirnov and Levene’s tests were first used to check for normality of the distribution and variance homogeneity of the sample residuals, respectively. In addition, linear regression analyses were conducted to determine the relationships between FFlT, LFlT, FFrT, LFrT, FlH and SLH. All statistical analyses were conducted with SPSS 19.0 software package (SPSS Inc., CHI, IL, USA).
species (Table 1, Fig. 1b). At the species level, mowing significantly prolonged LFlT of A. trullifolia var. linearis and C. scaposa by 5.6 days and 7.8 days, but did not change LFlT of T. farreri (Table 1, Fig. 1b). In addition, N addition and mowing interactively affected LFlT across all three species (Table 1). Mowing prolonged LFlT by 2.6 days under the N non-addition treatment, but prolonged it by 7.9 days under the N addition treatment (Fig. 1b).
3. Result
3.2. Fruiting phenology
3.1. Flowering phenology
N Addition did not significantly affect FFrT across the three species (Table 1). At the species level, N addition delayed FFrT of C. scaposa by 3.6 days, but did not affect FFrT of the other two species (Table 1, Fig. 1c). Mowing prolonged FFrT by 7.4 days across all three species (Table 1, Fig. 1c). Mowing prolonged FFrT of A. trullifolia var. linearis and C. scaposa by 7.7 and 11.3 days, respectively, but had no effect on T. farreri (Table 1, Fig. 1c). The interaction between N addition and mowing on FFrT was not significant (Table 1). N addition delayed LFrT by 3.5 days across all three species (Table 1). At the species level, N addition delayed LFrT of C. scaposa by 8.8 days (Table 1, Fig. 1d). Mowing prolonged LFrT by 4.8 days across the three species, and prolonged LFrT of A. trullifolia var. linearis, C. scaposa and T. farreri by 2.2, 6.4, and 5.8 days (Table 1, Fig. 1d), respectively. The interaction between N addition and mowing on LFlT was not detected (Table 1).
N addition did not alter FFlT across the three species, although it delayed the FFlT of A. trullifolia var. linearis and C. scaposa by 4.5 and 3.9 days, respectively (Table 1, Fig. 1a). Mowing prolonged FFlT by 7.9 days across all three species (Table 1, Fig. 1a). In particular, mowing prolonged FFlT of A. trullifolia var. linearis and C. scaposa by 11.7 days and 11.1 days (Table 1, Fig. 1a), respectively. In addition, there was a significant interaction between N addition and mowing (Table 1). Mowing prolonged FFlT by 4.7 days under the N non-addition treatment, but prolonged it by 11.2 days under the N addition treatment (Fig. 1a). N addition delayed LFlT of A. trullifolia var. linearis and C. scaposa by 3.6 days and 6.0 days, but prolonged LFlT of T. farreri by 3.4 days (Table 1, Fig. 1b). Mowing prolonged LFlT by 5.3 days across all three
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Fig. 2. Flowering height of three plant species (AT, Anemone trullifolia var. linearis; CS, Caltha scaposa; TF, Trollius farreri) among the four treatments (N-, nitrogen nonaddition; N+, nitrogen addition; M-, non-mowing; M+, mowing). Error bars are means ± SE.
Fig. 4. Relationships between flowering height (FlH) and standing litter height (SLH) at the species levels.
Fig. 3. Standing litter height (SLH) among the four treatments (N-, nitrogen nonaddition; N+, nitrogen addition; M-, non-mowing; M+, mowing). The different letters above the error bars denote the difference among treatments was statistically significant at the level of P = 0.05, as revealed by three-way ANOVA followed by LSD test for multiple comparisons, respectively. Error bars are means ± SE.
3.3. Flowering height N addition significantly increased FlH by 16.4% across the three species (Table 1, Fig. 2). At the species level, N addition significantly increased FlH of A. trullifolia var. linearis and C. scaposa by 29.9% and 31.1%, but did not affect FlH of T. farreri (Table 1, Fig. 2). Mowing decreased FlH by 38.8% across the three species, but the strength of the mowing effect varied with species (Table 1, Fig. 2). Specifically, mowing significantly reduced FlH of A. trullifolia var. linearis, C. scaposa, and T. farreri by 53.4, 39.9, and 26.5% (Table 1, Fig. 2), respectively. In addition, N addition and mowing interactively affected FlH (Table 1), i.e., N addition increased FlH by 24.06% under the non-mowing but not under the mowing treatments across the three species (Fig. 2). 3.4. Standing litter height N addition alone increased standing litter height (SLH) by 51.3%, whereas mowing decreased SLH by 75.7% (Table 1, Fig. 3). Moreover, N addition increased SLH by 66.5% in the non-mowing, but not the mowing treatments (Table 1, Fig. 3). In addition, there was a positive linear relationship between SLH and FlH for all three species (Fig. 4a–c). Positive linear relationships were also detected between FlH and phenological variables including FFlT, LFlT, FFrT, and FFrT for A. trullifolia var. linearis (Fig. 5a, d, g, j) and C. scaposa (Fig. 5b, e, h, k). For T. farreri, FlH was positively correlated with only LFlT (Fig. 5l) but not FFrT, LFrT or FF1T (Fig. 5c, f, i).
4. Discussion Our results show that N addition delayed plant reproductive phenology of early-flowering forb species by increasing standing litter height and flowering height in a Tibetan alpine meadow. Conversely, mowing for hay removed stand litter, reduced flowering height and prolonged plant productive phenology. Importantly, our results reveal that N addition dramatically delayed plant reproductive phenology in the non-mowing but not in the mowing plots. These results suggest that N addition and mowing interactively affected reproductive phenology of early-flowering plants in the Tibetan alpine meadow. N addition can delay, advance or neutrally affect plant flowering time (Cleland et al., 2006; Smith et al., 2012; Xia and Wan, 2013; Xi et al., 2015). Our study showed that N addition delayed both the first and the last flowering time of A. trullifolia var. linearis and C. scaposa, which is consistent with numerous studies addressing N addition effect on flowering time (Cleland et al., 2006; Xia and Wan, 2013; Xi et al., 2015). This effect of N addition can be largely attributed to the N addition-induced increase in standing litter height, which can mediate plant phenology (Vile et al., 2006; Rocha et al., 2008; Egawa et al., 2009). An increase in standing litter height can increase both reproductive (e.g. flowering height) and vegetative (e.g. stem height and leaf area) traits which were positively correlated with flowering time (Kahmen and Poschlod, 2002; Diaz et al., 2007; Klimesova et al., 2008; Rocha et al., 2008; Sun and Frelich, 2011). Flowering height is important for plants to gain light for flower development (Vile et al., 2006) and to attract pollinators (Galen and Cuba 2001; Vile et al., 2006). Higher standing litter can lead to higher plant height (Rocha et al., 2008), and higher plant height can delay plant phenology species need more time to complete the vegetative growth (Sun and Frelich, 2011). Moreover, in addition to the pathway via increasing the accumulation of stand litter, N addition may also directly increase plant reproduc-
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Fig. 5. Relationships between first flowering time (FFlT, a–c), last flowering time (LFlT, d–f), first fruiting time (FFrT, g–i), and last fruiting time (LFrT, j–l) and flowering height (FlH) at the species levels.
tive allocation (flowering height) by increasing nutrient availability (Cleland et al., 2006; Smith et al., 2012; Xia and Wan 2013; Zhang et al., 2013; Xi et al., 2015). In addition, large amounts of standing litter decreased soil temperature (Sharrow and Wright 1977; Wan et al., 2002). Low temperature can delay plant flowing time by reducing the gene expression related to flowering time(Song et al., 2013; Wolkovich et al., 2012; Fu et al., 2015). Conversely, possibly because the species T. farreri has a relatively higher flowering height (Fig. A. 2 in Supplementary material), the flowering time of T. farreri was not affected by the changes in standing litter. Interestingly, N addition advanced the last flowering time of T. farreri. This may be due to two possible reasons. First, N addition can accelerate flower bud development (Ferrante et al., 2013), thus the flower budding time will be earlier under an N addition treatment (Cleland et al., 2006). Second, N addition can stimulate plant photosynthesis, and increase the plant growth rate (Xia and Wan, 2008). If flowering phenology is size-dependent, the faster plant growth rate can lead to an earlier flowering time under an N addition treatment. N-induced delay of flowering time resulted in delay of fruiting time for A. trullifolia var. linearis and C. scaposa. Fruiting time is often determined by flowering time (Kudo, 1993; Ferrante et al., 2013). Nevertheless, flowering height may also partly explain the variation in fruiting time. Indeed, fruiting time was positively correlated with
flowering height, in accordance with previous studies (Kudo, 1993; Bolmgren and Cowan, 2008). This further indicates that the delay of fruiting time was due to the N-induced increase in standing litter. Notably, although the last flowering time of T. farreri was prolonged, the fruiting time of this species was not affected by N addition. This may be due to the postponed flower development under N addition treatment (Ferrante et al., 2013), which would lengthen the flowering duration, and thus offset the N addition induced advancement of flowering time. Our results showed that mowing prolonged the flowering and fruiting times for both A. trullifolia var. linearis and C. scaposa, which is consistent with many previous studies (Freeman et al., 2003; Trtikova, 2008). In contrast to facilitation effects of N deposition, mowing removed almost all standing litter (Jensen and Meyer, 2001; Wan et al., 2002; Bonanomi et al., 2009). Consequently, the plants spent much less energy on the flowering stems, resulting in an advance of flowering and fruiting times. In addition, because there was no standing litter, an increased temperature may be propitious to the flowering and fruiting of plants in cold conditions (Korner and Basler, 2010; Wolkovich et al., 2012). Moreover, mowing may also increase the photosynthetically active radiation, which would stimulate plant growth. Indeed, the plant size (vegetative height) in the mowing plots was larger than that in the non-mowing plots (Fig. A. 3 in Supplementary material). The fact
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that the advance of last fruiting time for the species T. farreri may be due to that higher flowering stems were more attractive to pollinators (Galen and Cuba, 2001; Vile et al., 2006). We indeed observed there were more pollinator insects visiting flowers of T. farreri in mowing plots (personal observations by Liu and Wu). Nevertheless, given that the effects of N addition on plant reproductive phenology were largely due to the increase in standing litter, removing standing litter should have partly eliminated the effects of N addition. 5. Conclusion Our results demonstrated both N addition and mowing for hay significantly changed the productive phenology of early-flowering plants by changing the accumulation of standing litter. Since there are few early-flowering species in the Tibetan alpine meadows and similar cold regions, changes in reproductive phenology (e.g. flowering time) may result in mismatches between animals (e.g. early pollinators and florivorous insects) and plants, lead to variations in seed production, and consequently alter community structure. Our study indicates that the effects of climate change on plant phenology can be mediated by grassland management strategies in the high density grasslands. Acknowledgements We are grateful to Guoyong Li, Junpeng Mu, Jian Feng, Junren Xian, and Juanjuan Han for field assistance and the suggestive comments on the early draft. This study was supported by the National Natural Science Foundation of China (31200375), China Postdoctoral Science Foundation (2012M520066, 2013T60699), and Outstanding Youth Training Foundation of Henan University (yqpy20140031). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ecoleng.2016.11. 033. References Bolmgren, K., Cowan, P.D., 2008. Time-size tradeoffs: a phylogenetic comparative study of flowering time, plant height and seed mass in a north-temperate flora. Oikos 117, 424–429. Bonanomi, G., Caporaso, S., Allegrezza, M., 2009. Effects of nitrogen enrichment, plant litter removal and cutting on a species-rich Mediterranean calcareous grassland. Plant Biosyst. 143, 443–455. Cao, S., Chen, L., Yu, X., 2009. Impact of China’s Grain for Green Project on the landscape of vulnerable arid and semi-arid agricultural regions: a case study in northern Shaanxi Province. J. Appl. Ecol. 46, 536–543. Cao, C., Jiang, S., Ying, Z., Zhang, F., Han, X., 2011. Spatial variability of soil nutrients and microbiological properties after the establishment of leguminous shrub Caragana microphylla Lam. plantation on sand dune in the Horqin Sandy Land of Northeast China. Ecol. Eng. 37, 1467–1475. Chuine, I., Yiou, P., Viovy, N., Seguin, B., Daux, V., Ladurie, E.L.R., 2004. Historical phenology: grape ripening as a past climate indicator. Nature 432, 289–290. Cleland, E.E., Chiariello, N.R., Loarie, S.R., Mooney, H.A., Field, C.B., 2006. Diverse responses of phenology to global changes in a grassland ecosystem. Proc. Nat. Acad. Sci. U. S. A. 103, 13740–13744. Diaz, S., Lavorel, S., McIntyre, S.U.E., Falczuk, V., Casanoves, F., Milchunas, D.G., Skarpe, C., Rusch, G., Sternberg, M., Noy-Meir, I., Landsberg, J., Zhang, W., Clark, H., Campbell, B.D., Landsberg, J., 2007. Plant trait responses to grazing-a global synthesis. Glob. Change Biol. 13, 313–341. Egawa, C., Koyama, A., Tsuyuzaki, S., 2009. Relationships between the developments of seedbank, standing vegetation and litter in a post-mined peatland. Plant Ecol. 203, 217–228. Encinas-Viso, F., Revilla, T.A., Etienne, R.S., 2012. Phenology drives mutualistic network structure and diversity. Ecol. Lett. 15, 198–208. Ferrante, A., Savin, R., Slafer, G.A., 2013. Floret development and grain setting differences between modern durum wheats under contrasting nitrogen availability. J. Exp. Bot. 64, 169–184.
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Effects of nitrogen addition and mowing on reproductive phenology of three early-flowering forb species in a Tibetan alpine meadow Yinzhan Liu a,1 , Renhui Miao a,1 , Auqun Chen a , Yuan Miao a , Yanjie Liu b , Xinwei Wu c,∗ a b c
International Joint Research Laboratory for Global Change Ecology, College of Life Sciences, Henan University, Kaifeng, Henan 475004, China Department of Biology, University of Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany Department of Ecology, College of Life Sciences, Nanjing University, Nanjing, Jiangsu, 210093, China
a r t i c l e
i n f o
Article history: Received 22 June 2016 Received in revised form 15 September 2016 Accepted 13 November 2016 Keywords: Flowering time Fruiting time Hay mowing Nitrogen deposition Qinghai-Tibetan plateau Standing litter
a b s t r a c t Nitrogen (N) deposition and land use are likely to alter plant phenology, with subsequent effects on community structure and ecosystem function. Phenological responses of three early-flowering species, Anemone trullifolia var. linearis, Caltha scaposa, and Trollius farreri to N addition and hay mowing were investigated in a Tibetan alpine meadow over three years. N addition significantly delayed the first flowering time of A. trullifolia var. linearis (by 11.7 days) and C. scaposa (by 11.1 days), but did not affect that of T. farreri. Mowing prolonged the first flowering time and first fruiting time by 4.7 and 7.4 days across all three species. Significant interactions between mowing and N addition on reproductive phenology characteristics were detected. Mowing induced changes in the first and last flowering times were 6.5 and 4.3 days earlier in the N addition plots than those in the N non-addition plots. The changes in reproductive time were mainly attributed to the variations in standing litter in each treatment, i.e. reproductive phenology (timing of flowering and fruiting) was positively associated with litter accumulation. Our results indicate that N deposition and land use can affect plant phenology by changing the accumulation of standing litter in Tibetan alpine meadows. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Plant phenology is the seasonal timing of environmentalmediated phenomenon, and therefore sensitive to environmental changes (Cleland et al., 2006; Neil and Wu, 2006; Wolkovich et al., 2012; Guo et al., 2013; Fu et al., 2015; Ge et al., 2015). Responses of plant phenology to environmental changes can alter community structure (Encinas-Viso et al., 2012), inter-species interactions (Rafferty and Ives, 2011), and ecosystem carbon exchange (Xia et al., 2015). Plant phenology is thus widely regarded as an important ˜ indicator of global change (Menzel and Fabian 1999; Penuelas and Filella 2001; Chuine et al., 2004; Keenan, 2015).
Abbrevations: FFlT, First flowering time; LFlT, last flowering time; FFrT, first fruiting time; LFrT, last fruiting time; AT, Anemone trullifolia var. linearis; CS, Caltha scaposa; TF, Trollius farreri. ∗ Corresponding author at: Department of Eology, College of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing, Jiangsu, 210023, China. E-mail address: [email protected] (X. Wu). 1 These authors contribute equally to this paper. http://dx.doi.org/10.1016/j.ecoleng.2016.11.033 0925-8574/© 2016 Elsevier B.V. All rights reserved.
As an important aspect of global change, nitrogen (N) deposition is regionally becoming serious (Galloway et al., 2004; Liu et al., 2013). N deposition has been demonstrated to facilitate plant photosynthesis (Fleischer et al., 2013) and stimulate plant growth (Xia and Wan, 2008; Cao et al., 2011; Lü et al., 2011; Zhang et al., 2013), which may further alter plant reproductive phenology (Cleland et al., 2006; Smith et al., 2012; Xia and Wan, 2013; Xi et al., 2015). For example, Cleland et al. (2006) reported that N addition prolonged flowering times of forbs but delayed those of grasses in an American annual grassland. Smith et al. (2012) found contrary results in an alpine tundra. Xia and Wan (2013) showed that N addition in a temperate steppe did not affect flowering time at the community level. Therefore, the effects of N deposition on plant reproductive phenology remain controversial across plant functional groups and species in different ecosystems. Mowing for hay is an important human activity in grazing pastures, and can change microclimates (Wan et al., 2002) and associated plant traits such as plant height and specific leaf area (Kahmen and Poschlod, 2002; Diaz et al., 2007; Klimesova et al., 2008), which are closely related to plant reproductive phenology (Sun and Frelich, 2011; Wolkovich et al., 2012). However, there is little evidence for the effects of mowing on plant reproductive
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phenology. We know only several cases in which mowing has been shown to advance plant flowering time to avoiding to be cut off because of the changes of selective forces during evolution (Reisch and Poschlod, 2008), or delay plant reproductive phenology by increasing the time to regenerate lost tissue (Freeman et al., 2003) and reduce the growth rate (Trtikova, 2008). Therefore, the effects of mowing on plant reproductive phenology are also unclear. The eastern part of the Tibetan plateau is characterized by extensive alpine meadows. This area has experienced universal N deposition (0.53 kg N ha −2 yr −1 ) in the last few years and it is predicted to consistently increase in the future (Liu et al., 2013). Meanwhile, these meadows provide large amounts of high-quality forages, supporting millions of livestock (Xiang et al., 2009). Mowing for hay is the only way for Chinese herdsman to store forages in winter (Cao et al., 2009). Mowing removes the aboveground part of the plant, which may alleviate the N induced changes in plant growth and alter associated plant reproductive phenology. Thus, N deposition and mowing might interactively affect plant reproductive phenology in these meadows. However, to the best of our knowledge, no study has examined the interaction between N addition and mowing in this area. In the present study, we conducted a manipulative 3-year field experiment involving N addition (addition vs. non-addition) and mowing (mowing vs. non-mowing) in a Tibetan alpine meadow. We quantified plant reproductive phenology traits including the first and last flowering times, the first and last fruiting times, and community standing litter height as well as flowering height of three early spring flowering species. These data were used to examine the effects of both N deposition and mowing on plant reproductive phenology of early-flowering forb species to predict responses of plant phenology to global change.
2. Materials and methods 2.1. Study site The study was performed in Hongyuan County (32◦ 48 N, 102◦ 33 E) of Sichuan province in the eastern Qinghai-Tibetan Plateau. The altitude is about 3500 m a.s.l. The climate is characterized by a short and cool spring, summer, and autumn and a long winter. Mean annual temperature is 0.9 ◦ C, with maximum and minimum monthly means being 10.9 ◦ C and −10.3 ◦ C in July and January, respectively. Mean annual precipitation is 690 mm, almost 80% of which occurs between May and August. The soil is classified as Mat Cry-gelic Cambisol (Chinese Soil Taxonomy Research Group 1995) with high soil organic carbon (250 g kg−1 ) but low total nitrogen (8 g kg −1 ) and phosphorus (5 mg kg −1 ) content at a depth of 0–20 cm (Wu et al., 2011). The study area is dominated by Blysmus sinocompressus, Carex enervis ssp. Chuanxibeiensis, Deschampsia caespitosa, Anemone trullifolia var. linearis, Potentilla anserina, Haplosphaera himalayensis, Aster alpinus and Gentiana formosa. In the growing season (late May to late September), the peak value of total community coverage is over 90% and average plant height is > 30 cm in late-August (Wu et al., 2011). In particular, Anemone trullifolia var. linearis, Caltha scaposa and Trollius farreri were the only three early-flowering perennial forb species in the study area (Liu et al., 2011), and occupied 22–61%, 0.23–1.1%, and 0.76–1.85% of the total biomass. The first flowering time of the three species happened from early May to early June, which was in the first two months of the snow-free season at the study site. The first fruiting time of A. trullifolia var. linearis, and C. scaposa occurred in late May, and the first fruiting time of T. farreri occurred in early August. We here focused on the responses of reproductive phenology of these three species to N addition and mowing.
2.2. Experimental design In a fenced area of 1 ha, we conducted an orthogonal two factortwo level experiment in which we crossed N addition (addition vs. non-addition) with mowing (mowing vs. non-mowing), yielding a total of four treatments: (i) neither N addition nor mowing (N-, M); (ii) N addition only (N+, M-); (iii) mowing only (N-, M + ); (iv) both N addition and mowing (N+, M + ). Each of these four treatments was replicated five times (n = 20 in total), each replicate was a 1.5 × 0.75 m2 plot. N (in the form of urea; 5 g N m−2 yr−1 ) was added to the nitrogen addition treatments in early April from 2008 to 2010. We cut and removed aboveground plant parts (leaving 5 cm) in early October after all phenological events were complete in the mowing treatments in both 2008 and 2009. In addition, soil temperature (5 cm underground) was measured in three plots for mowing verse non-mowing treatments using thermometers (model DS1921G, Maxim Integrated Products, Sunnyvale, California, USA). Over a 6-month duration (from January 19th to July 23rd, 2010), the mean daily temperature (over 24 h of measurements made every 30 min) in the mowing plots was 0.84 ◦ C higher than in the non-mowing plots (Fig. A. 1 in Supplementary material). 2.3. Standing litter The average standing litter height was calculated following the method by Liu et al. (2012). The number of each plant species (species level abundance) was investigated in a subplot of 0.5 × 0.5 m2 in each plot on 26th to 28th of August in 2009. The standing litter height of each species (from three individuals) was measured in each plot from 21th to 25th of March 2010. The community standing litter height was calculated using the following equation: SLH =
n
Hi ×
1
Ai
n
1
Ai
Where Hi and Ai stood for the standing litter height and the abundance of the species i, and n was the number of species. 2.4. Phenological observations Three flowering stems of each species were randomly taped in each plot and measured on 21th to 25th of March 2010. The flowering height (FlH) of each species was calculated as the mean value of the flowering stems. The number of flower and fruit (if any) of the three species in each plot was investigated over a 5-day interval from 1st April (before the earliest flowering time) to 27 August (after the latest fruiting time) in 2010. The first and last flowering/fruiting proportions (y) were calculated using a quadratic equation, following the protocols of Sun and Frelich (2011), i.e., y = ax2 + bx + c, where x is the Julian date. We defined the time when 10% and 90% of flower were blooming as the first flowering times (FFlT) and the last flowering times (LFlT), respectively. We defined the time when 10% and 90% of fruit were borne as the first fruiting times (FFrT) and the last fruiting times (LFrT), respectively. 2.5. Data analysis Three-way ANOVAs were employed to determine the effects of species, N addition and mowing on reproductive properties including FFlT, LFlT, FFrT, LFrT, and FlH. Two-way ANOVAs were performed to determine the effects of N addition and mowing on the species level reproductive properties including FFlT, LFlT, FFrT, LFrT, FlH, and the community level standing litter height (i.e., SLH). Once a significant effect was detected, post hoc LSD tests were used to further elucidate treatment differences. Where parametric tests
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Table 1 Results of ANOVAs showing the effects of mowing (M), nitrogen addition (N), species and their interactions on the first flowering times (FFlT), the last flowering times (LFlT), the first fruiting times (FFrT), the last fruiting times (LFrT), and flowering height (FlH) at the community level and the species level, df and F values are provided. ˆ , P < 0.1; * , P < 0.05; ** , P < 0.01; *** , P < 0.001. df
FFlT
LFlT
FFrT
LFrT
FlH
M N species M*N M * species N * species M * N * species
1 1 2 1 2 2 2
57.9*** 1.6 68.9*** 9.0** 10.8*** 8.4*** 0.6
17.7*** 2.6 262.6*** 4.3* 1.6 5.1* 0.5
41.8*** 2.0 105.6*** 0.1 4.0* 0.7 1.9
16.8*** 8.8** 172.5*** 1.9 1.3 5.4** 0.5
107.2*** 11.2** 102.2*** 6.7* 6.6** 1.9 0.6
Anemone trullifolia var. linearis M N M*N
1 1 1
44.6*** 5.3* 1.3
20.2*** 8.5* 0.5
23.6* 0.5 0.1
8.6** 1.8 0.1
51.6*** 7.2** 0.3
Caltha scaposa M N M*N
1 1 1
36.2*** 6.5* 2.1
6.3* 3.5ˆ 1.7
33.6*** 3.2ˆ 3.2ˆ
5.6* 11.0** 1.4
21.6*** 5.8* 4.3ˆ
Trollius farreri M N M*N
1 1 1
0.4 6.6* 6.6*
1.9 4.3ˆ 3.0
1.9 0.0 0.5
7.5* 0.1 0.5
34.4*** 0.3 5.5*
Fig. 1. First flowering times (FFlT, a), last flowering times (LFlT, b), first fruiting times (FFrT, c), and last fruiting times (LFrT, d) of three plant species (AT, Anemone trullifolia var. linearis; CS, Caltha scaposa; TF, Trollius farreri) among the four treatments (N-, nitrogen non-addition; N+, nitrogen addition; M-, non-mowing; M+, mowing). Error bars are means ± SE.
were performed, Kolmogorov-Smirnov and Levene’s tests were first used to check for normality of the distribution and variance homogeneity of the sample residuals, respectively. In addition, linear regression analyses were conducted to determine the relationships between FFlT, LFlT, FFrT, LFrT, FlH and SLH. All statistical analyses were conducted with SPSS 19.0 software package (SPSS Inc., CHI, IL, USA).
species (Table 1, Fig. 1b). At the species level, mowing significantly prolonged LFlT of A. trullifolia var. linearis and C. scaposa by 5.6 days and 7.8 days, but did not change LFlT of T. farreri (Table 1, Fig. 1b). In addition, N addition and mowing interactively affected LFlT across all three species (Table 1). Mowing prolonged LFlT by 2.6 days under the N non-addition treatment, but prolonged it by 7.9 days under the N addition treatment (Fig. 1b).
3. Result
3.2. Fruiting phenology
3.1. Flowering phenology
N Addition did not significantly affect FFrT across the three species (Table 1). At the species level, N addition delayed FFrT of C. scaposa by 3.6 days, but did not affect FFrT of the other two species (Table 1, Fig. 1c). Mowing prolonged FFrT by 7.4 days across all three species (Table 1, Fig. 1c). Mowing prolonged FFrT of A. trullifolia var. linearis and C. scaposa by 7.7 and 11.3 days, respectively, but had no effect on T. farreri (Table 1, Fig. 1c). The interaction between N addition and mowing on FFrT was not significant (Table 1). N addition delayed LFrT by 3.5 days across all three species (Table 1). At the species level, N addition delayed LFrT of C. scaposa by 8.8 days (Table 1, Fig. 1d). Mowing prolonged LFrT by 4.8 days across the three species, and prolonged LFrT of A. trullifolia var. linearis, C. scaposa and T. farreri by 2.2, 6.4, and 5.8 days (Table 1, Fig. 1d), respectively. The interaction between N addition and mowing on LFlT was not detected (Table 1).
N addition did not alter FFlT across the three species, although it delayed the FFlT of A. trullifolia var. linearis and C. scaposa by 4.5 and 3.9 days, respectively (Table 1, Fig. 1a). Mowing prolonged FFlT by 7.9 days across all three species (Table 1, Fig. 1a). In particular, mowing prolonged FFlT of A. trullifolia var. linearis and C. scaposa by 11.7 days and 11.1 days (Table 1, Fig. 1a), respectively. In addition, there was a significant interaction between N addition and mowing (Table 1). Mowing prolonged FFlT by 4.7 days under the N non-addition treatment, but prolonged it by 11.2 days under the N addition treatment (Fig. 1a). N addition delayed LFlT of A. trullifolia var. linearis and C. scaposa by 3.6 days and 6.0 days, but prolonged LFlT of T. farreri by 3.4 days (Table 1, Fig. 1b). Mowing prolonged LFlT by 5.3 days across all three
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Fig. 2. Flowering height of three plant species (AT, Anemone trullifolia var. linearis; CS, Caltha scaposa; TF, Trollius farreri) among the four treatments (N-, nitrogen nonaddition; N+, nitrogen addition; M-, non-mowing; M+, mowing). Error bars are means ± SE.
Fig. 4. Relationships between flowering height (FlH) and standing litter height (SLH) at the species levels.
Fig. 3. Standing litter height (SLH) among the four treatments (N-, nitrogen nonaddition; N+, nitrogen addition; M-, non-mowing; M+, mowing). The different letters above the error bars denote the difference among treatments was statistically significant at the level of P = 0.05, as revealed by three-way ANOVA followed by LSD test for multiple comparisons, respectively. Error bars are means ± SE.
3.3. Flowering height N addition significantly increased FlH by 16.4% across the three species (Table 1, Fig. 2). At the species level, N addition significantly increased FlH of A. trullifolia var. linearis and C. scaposa by 29.9% and 31.1%, but did not affect FlH of T. farreri (Table 1, Fig. 2). Mowing decreased FlH by 38.8% across the three species, but the strength of the mowing effect varied with species (Table 1, Fig. 2). Specifically, mowing significantly reduced FlH of A. trullifolia var. linearis, C. scaposa, and T. farreri by 53.4, 39.9, and 26.5% (Table 1, Fig. 2), respectively. In addition, N addition and mowing interactively affected FlH (Table 1), i.e., N addition increased FlH by 24.06% under the non-mowing but not under the mowing treatments across the three species (Fig. 2). 3.4. Standing litter height N addition alone increased standing litter height (SLH) by 51.3%, whereas mowing decreased SLH by 75.7% (Table 1, Fig. 3). Moreover, N addition increased SLH by 66.5% in the non-mowing, but not the mowing treatments (Table 1, Fig. 3). In addition, there was a positive linear relationship between SLH and FlH for all three species (Fig. 4a–c). Positive linear relationships were also detected between FlH and phenological variables including FFlT, LFlT, FFrT, and FFrT for A. trullifolia var. linearis (Fig. 5a, d, g, j) and C. scaposa (Fig. 5b, e, h, k). For T. farreri, FlH was positively correlated with only LFlT (Fig. 5l) but not FFrT, LFrT or FF1T (Fig. 5c, f, i).
4. Discussion Our results show that N addition delayed plant reproductive phenology of early-flowering forb species by increasing standing litter height and flowering height in a Tibetan alpine meadow. Conversely, mowing for hay removed stand litter, reduced flowering height and prolonged plant productive phenology. Importantly, our results reveal that N addition dramatically delayed plant reproductive phenology in the non-mowing but not in the mowing plots. These results suggest that N addition and mowing interactively affected reproductive phenology of early-flowering plants in the Tibetan alpine meadow. N addition can delay, advance or neutrally affect plant flowering time (Cleland et al., 2006; Smith et al., 2012; Xia and Wan, 2013; Xi et al., 2015). Our study showed that N addition delayed both the first and the last flowering time of A. trullifolia var. linearis and C. scaposa, which is consistent with numerous studies addressing N addition effect on flowering time (Cleland et al., 2006; Xia and Wan, 2013; Xi et al., 2015). This effect of N addition can be largely attributed to the N addition-induced increase in standing litter height, which can mediate plant phenology (Vile et al., 2006; Rocha et al., 2008; Egawa et al., 2009). An increase in standing litter height can increase both reproductive (e.g. flowering height) and vegetative (e.g. stem height and leaf area) traits which were positively correlated with flowering time (Kahmen and Poschlod, 2002; Diaz et al., 2007; Klimesova et al., 2008; Rocha et al., 2008; Sun and Frelich, 2011). Flowering height is important for plants to gain light for flower development (Vile et al., 2006) and to attract pollinators (Galen and Cuba 2001; Vile et al., 2006). Higher standing litter can lead to higher plant height (Rocha et al., 2008), and higher plant height can delay plant phenology species need more time to complete the vegetative growth (Sun and Frelich, 2011). Moreover, in addition to the pathway via increasing the accumulation of stand litter, N addition may also directly increase plant reproduc-
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Fig. 5. Relationships between first flowering time (FFlT, a–c), last flowering time (LFlT, d–f), first fruiting time (FFrT, g–i), and last fruiting time (LFrT, j–l) and flowering height (FlH) at the species levels.
tive allocation (flowering height) by increasing nutrient availability (Cleland et al., 2006; Smith et al., 2012; Xia and Wan 2013; Zhang et al., 2013; Xi et al., 2015). In addition, large amounts of standing litter decreased soil temperature (Sharrow and Wright 1977; Wan et al., 2002). Low temperature can delay plant flowing time by reducing the gene expression related to flowering time(Song et al., 2013; Wolkovich et al., 2012; Fu et al., 2015). Conversely, possibly because the species T. farreri has a relatively higher flowering height (Fig. A. 2 in Supplementary material), the flowering time of T. farreri was not affected by the changes in standing litter. Interestingly, N addition advanced the last flowering time of T. farreri. This may be due to two possible reasons. First, N addition can accelerate flower bud development (Ferrante et al., 2013), thus the flower budding time will be earlier under an N addition treatment (Cleland et al., 2006). Second, N addition can stimulate plant photosynthesis, and increase the plant growth rate (Xia and Wan, 2008). If flowering phenology is size-dependent, the faster plant growth rate can lead to an earlier flowering time under an N addition treatment. N-induced delay of flowering time resulted in delay of fruiting time for A. trullifolia var. linearis and C. scaposa. Fruiting time is often determined by flowering time (Kudo, 1993; Ferrante et al., 2013). Nevertheless, flowering height may also partly explain the variation in fruiting time. Indeed, fruiting time was positively correlated with
flowering height, in accordance with previous studies (Kudo, 1993; Bolmgren and Cowan, 2008). This further indicates that the delay of fruiting time was due to the N-induced increase in standing litter. Notably, although the last flowering time of T. farreri was prolonged, the fruiting time of this species was not affected by N addition. This may be due to the postponed flower development under N addition treatment (Ferrante et al., 2013), which would lengthen the flowering duration, and thus offset the N addition induced advancement of flowering time. Our results showed that mowing prolonged the flowering and fruiting times for both A. trullifolia var. linearis and C. scaposa, which is consistent with many previous studies (Freeman et al., 2003; Trtikova, 2008). In contrast to facilitation effects of N deposition, mowing removed almost all standing litter (Jensen and Meyer, 2001; Wan et al., 2002; Bonanomi et al., 2009). Consequently, the plants spent much less energy on the flowering stems, resulting in an advance of flowering and fruiting times. In addition, because there was no standing litter, an increased temperature may be propitious to the flowering and fruiting of plants in cold conditions (Korner and Basler, 2010; Wolkovich et al., 2012). Moreover, mowing may also increase the photosynthetically active radiation, which would stimulate plant growth. Indeed, the plant size (vegetative height) in the mowing plots was larger than that in the non-mowing plots (Fig. A. 3 in Supplementary material). The fact
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that the advance of last fruiting time for the species T. farreri may be due to that higher flowering stems were more attractive to pollinators (Galen and Cuba, 2001; Vile et al., 2006). We indeed observed there were more pollinator insects visiting flowers of T. farreri in mowing plots (personal observations by Liu and Wu). Nevertheless, given that the effects of N addition on plant reproductive phenology were largely due to the increase in standing litter, removing standing litter should have partly eliminated the effects of N addition. 5. Conclusion Our results demonstrated both N addition and mowing for hay significantly changed the productive phenology of early-flowering plants by changing the accumulation of standing litter. Since there are few early-flowering species in the Tibetan alpine meadows and similar cold regions, changes in reproductive phenology (e.g. flowering time) may result in mismatches between animals (e.g. early pollinators and florivorous insects) and plants, lead to variations in seed production, and consequently alter community structure. Our study indicates that the effects of climate change on plant phenology can be mediated by grassland management strategies in the high density grasslands. Acknowledgements We are grateful to Guoyong Li, Junpeng Mu, Jian Feng, Junren Xian, and Juanjuan Han for field assistance and the suggestive comments on the early draft. This study was supported by the National Natural Science Foundation of China (31200375), China Postdoctoral Science Foundation (2012M520066, 2013T60699), and Outstanding Youth Training Foundation of Henan University (yqpy20140031). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ecoleng.2016.11. 033. References Bolmgren, K., Cowan, P.D., 2008. Time-size tradeoffs: a phylogenetic comparative study of flowering time, plant height and seed mass in a north-temperate flora. Oikos 117, 424–429. Bonanomi, G., Caporaso, S., Allegrezza, M., 2009. Effects of nitrogen enrichment, plant litter removal and cutting on a species-rich Mediterranean calcareous grassland. Plant Biosyst. 143, 443–455. Cao, S., Chen, L., Yu, X., 2009. Impact of China’s Grain for Green Project on the landscape of vulnerable arid and semi-arid agricultural regions: a case study in northern Shaanxi Province. J. Appl. Ecol. 46, 536–543. Cao, C., Jiang, S., Ying, Z., Zhang, F., Han, X., 2011. Spatial variability of soil nutrients and microbiological properties after the establishment of leguminous shrub Caragana microphylla Lam. plantation on sand dune in the Horqin Sandy Land of Northeast China. Ecol. Eng. 37, 1467–1475. Chuine, I., Yiou, P., Viovy, N., Seguin, B., Daux, V., Ladurie, E.L.R., 2004. Historical phenology: grape ripening as a past climate indicator. Nature 432, 289–290. Cleland, E.E., Chiariello, N.R., Loarie, S.R., Mooney, H.A., Field, C.B., 2006. Diverse responses of phenology to global changes in a grassland ecosystem. Proc. Nat. Acad. Sci. U. S. A. 103, 13740–13744. Diaz, S., Lavorel, S., McIntyre, S.U.E., Falczuk, V., Casanoves, F., Milchunas, D.G., Skarpe, C., Rusch, G., Sternberg, M., Noy-Meir, I., Landsberg, J., Zhang, W., Clark, H., Campbell, B.D., Landsberg, J., 2007. Plant trait responses to grazing-a global synthesis. Glob. Change Biol. 13, 313–341. Egawa, C., Koyama, A., Tsuyuzaki, S., 2009. Relationships between the developments of seedbank, standing vegetation and litter in a post-mined peatland. Plant Ecol. 203, 217–228. Encinas-Viso, F., Revilla, T.A., Etienne, R.S., 2012. Phenology drives mutualistic network structure and diversity. Ecol. Lett. 15, 198–208. Ferrante, A., Savin, R., Slafer, G.A., 2013. Floret development and grain setting differences between modern durum wheats under contrasting nitrogen availability. J. Exp. Bot. 64, 169–184.
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