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Long term effect of nitrogen addition on understory community in a Chinese boreal forest
Science of the Total Environment 646 (2019) 989–995
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Long term effect of nitrogen addition on understory community in a Chinese boreal forest Aijun Xing a,b,1, Longchao Xu a,b,1, Haihua Shen a,b,⁎, Enzai Du c, Xiuyuan Liu a, Jingyun Fang a,b,d a
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China University of Chinese Academy of Sciences, Beijing 100049, China State Key Laboratory of Earth Surface Processes and Resource Ecology, Faculty of Geographical Science, Beijing Normal University, 100875 Beijing, China d Institute of Ecology, College of Urban and Environmental Sciences, and Key Laboratory for Earth Surface Processes of the Ministry of Education, Peking University, Beijing 100871, China b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• A N addition experiment was conducted in a Chinese boreal forest. • N addition significantly and negatively affected mosses cover. • N addition had no significant effect on vascular plants species richness but changed community composition. • The effect of N addition varied across functional groups and shifted overtime.
a r t i c l e
i n f o
Article history: Received 12 June 2018 Received in revised form 24 July 2018 Accepted 24 July 2018 Available online 25 July 2018 Editor: Jay Gan Keywords: Nitrogen deposition Understory plants Species richness Community composition Boreal forest
a b s t r a c t Increasing atmospheric nitrogen (N) deposition is an important driver of biodiversity change. By conducting an eight-year N addition experiment (0, 20, 50 and 100 kg N ha−1 yr−1), we investigated the long-term effect of simulated N deposition on understory species composition and richness in a boreal forest, northeast China. We found that moss cover decreased significantly with increasing N addition. N addition had no significant effect on vascular plants species richness but changed the plant community composition. The relative coverage of evergreen shrubs decreased, while that of graminoids increased under high-level N addition (100 kg N ha−1 yr−1). Under the high-level N treatment, Deyeuxia angustifolia cover increased significantly after 4 years, while that of Vaccinium vitis-idaea decreased significantly after 3 years and almost disappeared after 5 years. The negative effect of N addition on mosses and evergreen shrubs accumulated over time, while the positive effect on graminoids increased during the first 4 years and did not change significantly thereafter. Our results suggest that the effect of N deposition varies across functional groups and shifts over time. © 2018 Elsevier B.V. All rights reserved.
1. Introduction ⁎ Corresponding author at: State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. E-mail address: [email protected] (H. Shen). 1 These authors contributed equally to the work.
https://doi.org/10.1016/j.scitotenv.2018.07.350 0048-9697/© 2018 Elsevier B.V. All rights reserved.
Biodiversity is a key driver of ecosystem productivity (Duffy et al., 2017), and most of the biodiversity in forest ecosystems is contributed by understory plants (Gilliam, 2006, Gilliam, 2007). Increasing nitrogen (N) deposition is one of the major drivers that cause biodiversity loss
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Table 1 Repeated measures ANOVA results. Source Vascular plants total cover Treatment Error: plot Year Treatment ∗ year Error: Plot ∗ year Total Moss cover Treatment Error: plot Year Treatment ∗ year Error: plot ∗ year Total Richness (species/m2) Treatment Error: plot Year Treatment ∗ year Error: Plot ∗ year Total
df
SS
MS
F
p
3 8 7 21 56 95
6380 7740 11,090 2344 4543
2126.7 967.5 1584.3 111.6 81.1
2.20
0.17
19.53 1.38
b0.001 0.17
3 8 7 21 56 95
13,625 7129 11,305 5852 4386
4542 891 1615.1 278.6 78.3
5.10
0.03
20.62 3.56
b0.001 b0.001
3 8 7 21 56 95
42.59 73.86 37.57 22.72 30.06
14.20 9.23 5.37 1.08 0.54
1.54
0.28
10.00 2.02
b0.001 0.02
Bold values are significant at P b 0.05.
(Sala et al., 2000), as is consistently shown by both observational and experimental research (Suding et al., 2005; Bobbink et al., 2010; De Schrijver et al., 2011; Verheyen et al., 2012; Clark et al., 2013; Simkin et al., 2016). In China, N deposition has been dramatically increasing for decades (Liu et al., 2013), and the forests have been receiving high levels of N deposition (Du et al., 2014a). However, the long-term effects of N deposition on the plant diversity in China's forests have rarely been reported, especially regarding sensitive ecosystems such as boreal forests (Bobbink et al., 2010). Many previous studies have revealed the impacts of N deposition on understory biodiversity in forests, but the responses of understory plants to N addition are not consistent. For example, long-term N addition caused species loss in a temperate forest (Walter et al., 2017) and a tropical forest (Lu et al., 2010), while most studies found no overall effect of N fertilization on biodiversity but rather a change of community composition (Hurd et al., 1998; He and Barclay, 2000; Strengbom et al., 2001; Ostertag and Verville, 2002; Nordin et al., 2009). These inconsistencies might be attributed to differences in background N deposition, vegetation type and experiment duration (Bobbink et al., 2010; De Schrijver et al., 2011; Hedwall et al., 2013a; Gilliam et al., 2016). The homogeneity hypothesis assumes that the dominance of nitrophilic species will increase following increasing N input, while the abundance of N efficient species will decrease, resulting in changes in community structure and decreased diversity (Gilliam, 2006; Gilliam et al., 2016).
Fig. 1. Species richness and diversity in response to N addition over time (a, species richness; b, Shannon-Wiener index; and c, Pielou's index).
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Mosses are sensitive to N deposition, and declines in cover, frequency or biomass following N addition have been reported in boreal forests (Hallbäcken and Zhang, 1998; Makipaa, 1998; Nordin et al., 2009; Du et al., 2014b), temperate forests (Thomas et al., 1999) and tropical montane forests (Ostertag and Verville, 2002). Vascular plants of various functional groups have been shown to respond differently to N addition (Hallbäcken and Zhang, 1998; van Dobben et al., 1999; He and Barclay, 2000; Strengbom et al., 2002; Turkington et al., 2002; Lu et al., 2010), including an increase of fast-growing herbs and a decrease of slowgrowing dwarf shrubs (van Dobben et al., 1999; Strengbom et al., 2002; Turkington et al., 2002; Strengbom and Nordin, 2012). The hierarchical-response framework (HRF) suggests that the plant community response to chronic environmental change could either be linear or nonlinear, with individuals reacting firstly, followed by species reordering and finally species loss or immigration (Smith et al., 2009). Whole watershed fertilization of the Fernow Experimental Forest, one of the longest ongoing forest N addition experiments, found that the understory plant community did not respond after the first 6 years of N application (Gilliam et al., 1994; Gilliam et al., 2006), but afterward, a divergence occurred in community composition between the fertilized and unfertilized watersheds (Gilliam et al., 2016). Three-year N additions in a boreal forest significantly increased the abundance of the graminoid Deschampsia flexuosa (Strengbom et al., 2002), but its abundance decreased after 4 years of fertilization (Nordin et al., 2009). These studies emphasize the value of long-term experiments to capture the trajectory of plant community change. Due to prevailing N limitation, boreal forests have been evidenced to be highly sensitive to enhanced N deposition (Bobbink et al., 2010). In a boreal forest in Northeast China, three-year N addition exerted no significant effect on the understory species richness, but an obvious shift in species composition occurred (Du, 2017). However, the longterm effects of N deposition on the understory community of this boreal forest remain unclear. In this study, we report the results of an eight-year N addition experiment in the boreal forest to explore the effect of long-term simulated N deposition on understory community structure and biodiversity. Specifically, we aimed to address the following questions: 1) How do understory plants respond to 8-year N
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addition, and what is the sensitivity of understory plants? 2) Does 8 years N addition change community composition and cause species loss? 2. Materials and methods 2.1. Site description and experimental design The study site is located at the National Field Research Station of the Daxing'anling Forest Ecosystem (50°56′N, 121°30′E) in the Great Khingan Mountains, Northeast China. The elevation at the site is 825 m. The mean annual temperature (MAT) is −5.4 °C, and the mean annual precipitation (MAP) is 481 mm. The soil is brown coniferous forest soil (Du et al., 2013). The canopy layer is dominated by Larix gmelinii, and the understory plants is mainly consist of the deciduous shrubs Ledum palustre and Vaccinium uliginosum, the evergreen shrubs Rhododendron lapponicum and Vaccinium vitis-idaea, and the graminoid Deyeuxia angustifolia. The moss layer is mainly composed of Rhytidium rugosum and Aulacomnium palustre (see Table S1 for a list of understory plants). An N addition experiment was initiated in 2010. We selected a plain area and adopted a 4 treatment × 3 replicate randomized block design with a total of 12 plots. Each plot was 20 × 20 m2, and a 10-m buffering zone was established. The treatments were as follow: 0 (control), 20 kg N ha−1 yr−1 (Low N), 50 kg N ha−1 yr−1 (Medium N) and 100 kg N ha−1 yr−1 (High N). We sprayed dissolved ammonium nitrate with a backpack sprayer during May to September, and the control plot received equal amounts of water (details can be found in Du et al., 2013). 2.2. Vegetation investigation and soil chemistry analysis A vegetation investigation was conducted in July–August of each year. We established a 2.5-m buffer zone in each plot, and the entire survey was conducted within the 15 × 15 m2 area. Five permanent 1 × 1 m2 subplots were established: one in each corner and one in the center of the sampling area. We recorded the total number of all the vascular plants b1 m in height as the species richness and visually estimated the cover
Fig. 2. Plant cover changes over time under N addition (a, vascular plants total cover; and b, moss cover).
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of each species. In each subplot, we also estimated moss cover since mosses play a major role in boreal forests (Cornelissen et al., 2007). We collected soil samples from the organic and mineral layers (0–10 cm) from each corner and the center of each plot using a 5-cmdiameter auger in August 2016. Soil inorganic N and soil pH were determined. To measure soil inorganic N, we sieved soil samples through a 2mm sieve and extracted samples with 2 mol L−1 KCl. The extracts were then stored in a freezer for later analysis with a continuous flow analyzer (SEAL AA3, Germany). Soil pH was determined with a meter (Mettler Toledo, Switzerland) which was calibrated once every five records. 2.3. Statistical analysis Shannon-Wiener and Pielou's diversity index values were calculated based on species cover (Hill, 1973). We used repeated measures ANOVA to investigate the effect of treatment, year and their interaction on vascular plant total cover and species richness. Year was treated as a within group variable; treatment was treated as a between group variable; and plot was treated as a repeated measurement subject. Mauchly's test was used to assess the sphericity assumption. One-way ANOVA was used to investigate the treatment effect on soil ammonium, soil nitrate, total inorganic N and soil pH, with block as random effect. We used Tukey's HSD test for post hoc analysis. We estimated the sensitivity, which was determined as the year when a significant treatment effect became apparent, and the magnitude of the treatment effect. For the first analysis, we treated N dosage as a continuous variable and regressed it against moss cover, vascular plant total cover and the cover of different functional groups. To determine the magnitude of the treatment effect,
experiment (Fig. S2). For moss cover, the response ratio of the low N treatment to the control did not differ from 0, while that of the high N treatment to the control was b0 and showed a decreasing trend over time (Fig. 3a). Deciduous shrubs did not show a response to N addition (Fig. 3b), while the response ratio of evergreen shrubs began to fall below 0 in 2012 and showed a decreasing trend over time under high N treatment (Fig. 3c). The response ratio of forbs remained constant over time (Fig. 3d), while that of graminoid cover began to exceed 0 in 2011 under high N addition and increased until 2013 (Fig. 3e). The cover of the graminoid D. angustifolia started to increase under high N addition in 2013, and the trend continued (Fig. 4a). The cover of the deciduous shrub L. palustre started to decrease in 2014 under high N treatment (Fig. 4b), while the deciduous shrub V. uliginosum showed no obvious trend in response to N addition (Fig. 4c). The cover of the evergreen shrub V. vitis-idaea started to decline under high N relative to the control in 2012, and it became significantly lower under the high N treatment than the control treatment thereafter and almost disappeared from the plot in 2014 (Fig. 4d). 4. Discussion 4.1. The effect of simulated N deposition varied across functional groups In this study, we investigated the effects of simulated N deposition on plant diversity and community structure based on an 8-year N
Treatment we calculated the effect size as LnRR ¼ Ln Cover CoverControl
Statistical analyses were performed with R 3.3.1 (R Core Team, 2016). Diversity indexes were calculated with the Vegan package (Jari Oksanen et al., 2017). We conducted the repeated measures ANOVA with the package EZ (Lawrence, 2016) and performed the multiple comparisons with the Multcomp package (Hothorn et al., 2008). 3. Results 3.1. Effects of N addition across functional types Repeated measures ANOVA indicated no significant effect of N addition on understory species richness (Table 1 and Fig. 1). Furthermore, N addition had no significant effect on vascular plant total cover (Table 1 and Fig. 2a), but it significantly and negatively affected mosscover (Table 1 and Fig. 2b). The relative proportions of the different functional groups of plants in the vascular plant total cover varied over time. Under high N addition, the proportion of evergreen shrubs decreased over time from 66.4% (33.10 ± 11.46) in 2010 to 0.6% (0.2 ± 0.1) in 2017, but even under low N treatment, it decreased from 66.4% (29.93 ± 5.91) in 2010 to 32.4% (10.93 ± 1.07) in 2017, while the proportion remained relatively constant in the control plot. In contrast, the proportion of graminoids increased from 2.7% (1.33 ± 0.54) in 2010 to 59.5% (18.93 ± 11.75) in 2017 under high N addition. The proportions of deciduous shrubs and forbs remained approximately stable. 3.2. Temporal changes in the effects of N addition Diversity under high N treatment showed a decreasing trend (Table 1 and Fig. 1), and richness declined significantly under high N treatment over time (Fig. S1; r2 = 0.423, P b 0.001). Plants sensitivity to N addition varied. Moss cover was significantly and negatively related to N dose in the second year as well as in the subsequent years, and vascular plants total cover showed a decreasing trend with a significant effect observed only in 2014 (Fig. S2). Evergreen shrub cover started to show a negative trend in the second year of the
Fig. 3. Changes in the response ratios of plant cover to N addition during the 8-year study period (a, mosses; b, deciduous shrubs; c, evergreen shrubs; d, forbs; e, graminoids).
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Fig. 4. Effect of N addition on common species over time (a, Deyeuxia angustifolia; b, Ledum palustre; c, Vaccinium uliginosum; and d, Vaccinium vitis-idaea. “*” indicates a significant difference between the high N and control treatments at P b 0.05 as revealed by one way ANOVA in each year).
addition experiment. N addition did not affect species richness, but a decreasing trend was observed under high N addition (Fig. S2), which is consistent with a synthesis of results for boreal forests (Bobbink, 2004). Species richness was not affected but community composition changed (Fig. 3), which was consistent with HRF that plant community under chronic environmental changes may experience a community composition change firstly and finally species loss or immigration (Smith et al., 2009). Our results showed that N addition did not significantly affect vascular plants total cover but significantly decreased moss cover (Table 1 and Fig. 1). Mosses lack a specialized cuticle structure to regulate water or nutrient uptake (Hallbäcken and Zhang, 1998; Makipaa, 1998), and when N deposition is low (e.g. b12 kg N ha−1 yr−1), mosses might take up N at the expense of woody plants (Gundale et al., 2011; Gundale et al., 2014). However, moss N uptake will decrease with increasing N deposition rates (e.g. N12 kg N ha−1 yr−1, Gundale et al., 2014) and moss abundance will decline under high N deposition due to direct NH+ 4 toxicity or competition for light (Nordin et al., 2005; Bret-Harte et al., 2008; Gundale et al., 2014;), which make mosses more vulnerable than vascular plants to increased N deposition (Bobbink et al., 2010; Gundale et al., 2011; Pardo et al., 2011; Gundale et al., 2014). In our current study, N addition significantly increased soil nitrate and total inorganic N (Fig. 5), which might cause a decrease
in moss cover, and this decrease might be partly explained by light competition considering the uneven increase in D. angustifolia (Fig. 4). Vascular plants of different functional groups responded differently to N addition. The relative proportions of deciduous shrubs and forbs were approximately constant (Fig. 3). However, the relative proportion of evergreen shrubs (particularly V. vitis-idaea, Fig. 4d) decreased, while that of graminoids increased (Fig. 3). Deciduous shrubs and graminoids which share common response traits (e.g. generating additional meristems), were good competitors under fertilization; however evergreen shrubs were not (Bret-Harte et al., 2008). Soil inorganic N increased in the organic layer but soil pH did not respond to N addition (Fig. 5). As suggested by the homogeneity hypothesis, this improvement of N availability might have affected interspecific competition and caused a change in community composition (Gilliam et al., 2006, Gilliam et al., 2016). Plants adapted to low N availability, for example, ericaceous dwarf shrubs, investigate their biomass to well-defended leaves, which could slow their growth rate (Cornelissen et al., 2001); as inorganic N availability increased, they failed to compete with fastgrowing graminoids (Cornelissen et al., 2001; Gilliam, 2006; Bobbink et al., 2010). In addition, light often interacts with N fertilization to regulate plant community dynamics (Hautier et al., 2009; Hofmeister et al., 2009; Walter et al., 2016), and this factor might have contributed to the decrease of dwarf shrub V. vitis-idaea. A consistent negative N effect on
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Fig. 5. Effects of N addition on soil chemistry (a, soil ammonium; b, soil nitrate; c, total inorganic nitrogen; and d, soil pH. Bars with the same letter are not significantly different among treatments at P b 0.05).
V. vitis-idaea was found in our study and other fertilization experiments (Strengbom et al., 2001; Manninen et al., 2009), as well as field observations along an N deposition gradient (Strengbom et al., 2003) and this legacy might last long after the termination of N addition (Strengbom et al., 2001). V. vitis-idaea is recognized as a keystone species in boreal forests (Nordin et al., 2005; Hedwall et al., 2013b).This negative N fertilization effect can significantly affect aboveground biomass production and, consequently, the carbon cycle. 4.2. The effect of simulated N deposition changed over long time Understory plants in boreal forests responded rapidly to N fertilization, within one to 3 years (Makipaa, 1998; Strengbom et al., 2002; Du et al., 2014b), partly due to low background N deposition (Gilliam et al., 2016). However, the response of the plant community might exhibit temporal variation with a longer timespan (Nordin et al., 2009; Gilliam et al., 2016). The negative effect of simulated N deposition on mosses and evergreen shrubs accumulated over time, while the positive effect on graminoids increased during the first 4 years but did not change significantly thereafter (Fig. 3). Mosses and evergreen shrubs have been reported to be sensitive and react rapidly to N deposition (Makipaa, 1998; Ostertag and Verville, 2002; Du, 2017). Previous study at our site showed that the biomass of the moss R. rugosum exhibited a decreasing trend after 3 years fertilization (Du et al., 2014b), but this accumulated N deposition might eventually restructure the moss community (Strengbom et al., 2001). A combination of removal and fertilization experiments showed that loss of mosses and evergreen shrubs were compensated by graminoids and deciduous shrubs (Bret-Harte et al., 2008), and a slow recovery of the boreal forest plant community from N fertilization was reported (Strengbom et al., 2001). The most notable changes in vascular plant cover over time under high-level N treatment have been a decrease of the evergreen shrub V. vitis-idaea and increase of the grass D. angustifolia (Fig. 4). D. angustifolia is a mesic nitrophilic plant. A previous study in a tundra
ecosystem found it to be an invasive plant whose abundance increased following N addition, and it tended to be the dominant species in the community (Jin et al., 2016; Zong et al., 2016). The cover of the grass D. angustifolia increased under high-level N, but no increase was observed after 4 years. Similarly, previous studies have shown that the abundance of the grass Deschampsia flexuosa increased linearly with fertilization during the initial 3 years (Strengbom et al., 2002); however, this increase continued for another 3 years and then decreased and remained constant in the following years (Nordin et al., 2009). Variation in growing season weather conditions might interact with N fertilization to affect the plant community (Nordin et al., 2009). For example, a previous study documented that enhanced precipitation affects forbs and grasses differently, and N and precipitation interaction were observed (Zavaleta et al., 2003). This interaction was apparent in 2013 (Fig. 2; Fig. 4), especially for D. angustifolia (Fig. 4a), when extreme growing season precipitation occurred. D. angustifolia cover increased considerably in 2013 under high-level N addition and remained higher thereafter (Fig. 4). However, this N and precipitation interaction was not observed for mosses and V. vitis-idaea, which implies that these plants might hard to recover from negative N fertilization effect. 5. Conclusion Overall, the response of the plant community to 8 years of N addition in an old-growth boreal forest was consistent with the HRF framework and homogeneity hypothesis. N addition did not affect species richness but changed the community composition. Different plant functional groups exhibited varied responses to experimental N addition, and mosses were the most vulnerable. The relative proportion of evergreen shrubs in the vascular cover decreased, while that of graminoids increased under high-level N addition. D. angustifolia cover increased significantly after 4 years, while that of V. vitis-idaea decreased significantly after 3 years and almost disappeared after 5 years. The mechanisms underlying long term community dynamics following increased N deposition remain complex. Future experiments with multiple factors will
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Regional differences in the occurrence of understorey species reflect nitrogen deposition in Swedish forests. Ambio 32, 91–97. Suding, K.N., Collins, S.L., Gough, L., Clark, C., Cleland, E.E., Gross, K.L., et al., 2005. Functional- and abundance-based mechanisms explain diversity loss due to N fertilization. Proc. Natl. Acad. Sci. U. S. A. 102, 4387–4392. Thomas, S.C., Halpern, C.B., Falk, D.A., Liguori, D.A., Austin, K.A., 1999. Plant diversity in managed forests: understory responses to thinning and fertilization. Ecol. Appl. 9, 864–879. Turkington, R., John, E., Watson, S., Seccombe-Hett, P., 2002. The effects of fertilization and herbivory on the herbaceous vegetation of the boreal forest in north-western Canada: a 10-year study. J. Ecol. 90, 325–337. van Dobben, H.F., ter Braak, C.J.F., Dirkse, G.M., 1999. Undergrowth as a biomonitor for deposition of nitrogen and acidity in pine forest. For. Ecol. Manag. 114, 83–95. 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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Long term effect of nitrogen addition on understory community in a Chinese boreal forest Aijun Xing a,b,1, Longchao Xu a,b,1, Haihua Shen a,b,⁎, Enzai Du c, Xiuyuan Liu a, Jingyun Fang a,b,d a
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China University of Chinese Academy of Sciences, Beijing 100049, China State Key Laboratory of Earth Surface Processes and Resource Ecology, Faculty of Geographical Science, Beijing Normal University, 100875 Beijing, China d Institute of Ecology, College of Urban and Environmental Sciences, and Key Laboratory for Earth Surface Processes of the Ministry of Education, Peking University, Beijing 100871, China b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• A N addition experiment was conducted in a Chinese boreal forest. • N addition significantly and negatively affected mosses cover. • N addition had no significant effect on vascular plants species richness but changed community composition. • The effect of N addition varied across functional groups and shifted overtime.
a r t i c l e
i n f o
Article history: Received 12 June 2018 Received in revised form 24 July 2018 Accepted 24 July 2018 Available online 25 July 2018 Editor: Jay Gan Keywords: Nitrogen deposition Understory plants Species richness Community composition Boreal forest
a b s t r a c t Increasing atmospheric nitrogen (N) deposition is an important driver of biodiversity change. By conducting an eight-year N addition experiment (0, 20, 50 and 100 kg N ha−1 yr−1), we investigated the long-term effect of simulated N deposition on understory species composition and richness in a boreal forest, northeast China. We found that moss cover decreased significantly with increasing N addition. N addition had no significant effect on vascular plants species richness but changed the plant community composition. The relative coverage of evergreen shrubs decreased, while that of graminoids increased under high-level N addition (100 kg N ha−1 yr−1). Under the high-level N treatment, Deyeuxia angustifolia cover increased significantly after 4 years, while that of Vaccinium vitis-idaea decreased significantly after 3 years and almost disappeared after 5 years. The negative effect of N addition on mosses and evergreen shrubs accumulated over time, while the positive effect on graminoids increased during the first 4 years and did not change significantly thereafter. Our results suggest that the effect of N deposition varies across functional groups and shifts over time. © 2018 Elsevier B.V. All rights reserved.
1. Introduction ⁎ Corresponding author at: State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. E-mail address: [email protected] (H. Shen). 1 These authors contributed equally to the work.
https://doi.org/10.1016/j.scitotenv.2018.07.350 0048-9697/© 2018 Elsevier B.V. All rights reserved.
Biodiversity is a key driver of ecosystem productivity (Duffy et al., 2017), and most of the biodiversity in forest ecosystems is contributed by understory plants (Gilliam, 2006, Gilliam, 2007). Increasing nitrogen (N) deposition is one of the major drivers that cause biodiversity loss
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Table 1 Repeated measures ANOVA results. Source Vascular plants total cover Treatment Error: plot Year Treatment ∗ year Error: Plot ∗ year Total Moss cover Treatment Error: plot Year Treatment ∗ year Error: plot ∗ year Total Richness (species/m2) Treatment Error: plot Year Treatment ∗ year Error: Plot ∗ year Total
df
SS
MS
F
p
3 8 7 21 56 95
6380 7740 11,090 2344 4543
2126.7 967.5 1584.3 111.6 81.1
2.20
0.17
19.53 1.38
b0.001 0.17
3 8 7 21 56 95
13,625 7129 11,305 5852 4386
4542 891 1615.1 278.6 78.3
5.10
0.03
20.62 3.56
b0.001 b0.001
3 8 7 21 56 95
42.59 73.86 37.57 22.72 30.06
14.20 9.23 5.37 1.08 0.54
1.54
0.28
10.00 2.02
b0.001 0.02
Bold values are significant at P b 0.05.
(Sala et al., 2000), as is consistently shown by both observational and experimental research (Suding et al., 2005; Bobbink et al., 2010; De Schrijver et al., 2011; Verheyen et al., 2012; Clark et al., 2013; Simkin et al., 2016). In China, N deposition has been dramatically increasing for decades (Liu et al., 2013), and the forests have been receiving high levels of N deposition (Du et al., 2014a). However, the long-term effects of N deposition on the plant diversity in China's forests have rarely been reported, especially regarding sensitive ecosystems such as boreal forests (Bobbink et al., 2010). Many previous studies have revealed the impacts of N deposition on understory biodiversity in forests, but the responses of understory plants to N addition are not consistent. For example, long-term N addition caused species loss in a temperate forest (Walter et al., 2017) and a tropical forest (Lu et al., 2010), while most studies found no overall effect of N fertilization on biodiversity but rather a change of community composition (Hurd et al., 1998; He and Barclay, 2000; Strengbom et al., 2001; Ostertag and Verville, 2002; Nordin et al., 2009). These inconsistencies might be attributed to differences in background N deposition, vegetation type and experiment duration (Bobbink et al., 2010; De Schrijver et al., 2011; Hedwall et al., 2013a; Gilliam et al., 2016). The homogeneity hypothesis assumes that the dominance of nitrophilic species will increase following increasing N input, while the abundance of N efficient species will decrease, resulting in changes in community structure and decreased diversity (Gilliam, 2006; Gilliam et al., 2016).
Fig. 1. Species richness and diversity in response to N addition over time (a, species richness; b, Shannon-Wiener index; and c, Pielou's index).
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Mosses are sensitive to N deposition, and declines in cover, frequency or biomass following N addition have been reported in boreal forests (Hallbäcken and Zhang, 1998; Makipaa, 1998; Nordin et al., 2009; Du et al., 2014b), temperate forests (Thomas et al., 1999) and tropical montane forests (Ostertag and Verville, 2002). Vascular plants of various functional groups have been shown to respond differently to N addition (Hallbäcken and Zhang, 1998; van Dobben et al., 1999; He and Barclay, 2000; Strengbom et al., 2002; Turkington et al., 2002; Lu et al., 2010), including an increase of fast-growing herbs and a decrease of slowgrowing dwarf shrubs (van Dobben et al., 1999; Strengbom et al., 2002; Turkington et al., 2002; Strengbom and Nordin, 2012). The hierarchical-response framework (HRF) suggests that the plant community response to chronic environmental change could either be linear or nonlinear, with individuals reacting firstly, followed by species reordering and finally species loss or immigration (Smith et al., 2009). Whole watershed fertilization of the Fernow Experimental Forest, one of the longest ongoing forest N addition experiments, found that the understory plant community did not respond after the first 6 years of N application (Gilliam et al., 1994; Gilliam et al., 2006), but afterward, a divergence occurred in community composition between the fertilized and unfertilized watersheds (Gilliam et al., 2016). Three-year N additions in a boreal forest significantly increased the abundance of the graminoid Deschampsia flexuosa (Strengbom et al., 2002), but its abundance decreased after 4 years of fertilization (Nordin et al., 2009). These studies emphasize the value of long-term experiments to capture the trajectory of plant community change. Due to prevailing N limitation, boreal forests have been evidenced to be highly sensitive to enhanced N deposition (Bobbink et al., 2010). In a boreal forest in Northeast China, three-year N addition exerted no significant effect on the understory species richness, but an obvious shift in species composition occurred (Du, 2017). However, the longterm effects of N deposition on the understory community of this boreal forest remain unclear. In this study, we report the results of an eight-year N addition experiment in the boreal forest to explore the effect of long-term simulated N deposition on understory community structure and biodiversity. Specifically, we aimed to address the following questions: 1) How do understory plants respond to 8-year N
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addition, and what is the sensitivity of understory plants? 2) Does 8 years N addition change community composition and cause species loss? 2. Materials and methods 2.1. Site description and experimental design The study site is located at the National Field Research Station of the Daxing'anling Forest Ecosystem (50°56′N, 121°30′E) in the Great Khingan Mountains, Northeast China. The elevation at the site is 825 m. The mean annual temperature (MAT) is −5.4 °C, and the mean annual precipitation (MAP) is 481 mm. The soil is brown coniferous forest soil (Du et al., 2013). The canopy layer is dominated by Larix gmelinii, and the understory plants is mainly consist of the deciduous shrubs Ledum palustre and Vaccinium uliginosum, the evergreen shrubs Rhododendron lapponicum and Vaccinium vitis-idaea, and the graminoid Deyeuxia angustifolia. The moss layer is mainly composed of Rhytidium rugosum and Aulacomnium palustre (see Table S1 for a list of understory plants). An N addition experiment was initiated in 2010. We selected a plain area and adopted a 4 treatment × 3 replicate randomized block design with a total of 12 plots. Each plot was 20 × 20 m2, and a 10-m buffering zone was established. The treatments were as follow: 0 (control), 20 kg N ha−1 yr−1 (Low N), 50 kg N ha−1 yr−1 (Medium N) and 100 kg N ha−1 yr−1 (High N). We sprayed dissolved ammonium nitrate with a backpack sprayer during May to September, and the control plot received equal amounts of water (details can be found in Du et al., 2013). 2.2. Vegetation investigation and soil chemistry analysis A vegetation investigation was conducted in July–August of each year. We established a 2.5-m buffer zone in each plot, and the entire survey was conducted within the 15 × 15 m2 area. Five permanent 1 × 1 m2 subplots were established: one in each corner and one in the center of the sampling area. We recorded the total number of all the vascular plants b1 m in height as the species richness and visually estimated the cover
Fig. 2. Plant cover changes over time under N addition (a, vascular plants total cover; and b, moss cover).
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of each species. In each subplot, we also estimated moss cover since mosses play a major role in boreal forests (Cornelissen et al., 2007). We collected soil samples from the organic and mineral layers (0–10 cm) from each corner and the center of each plot using a 5-cmdiameter auger in August 2016. Soil inorganic N and soil pH were determined. To measure soil inorganic N, we sieved soil samples through a 2mm sieve and extracted samples with 2 mol L−1 KCl. The extracts were then stored in a freezer for later analysis with a continuous flow analyzer (SEAL AA3, Germany). Soil pH was determined with a meter (Mettler Toledo, Switzerland) which was calibrated once every five records. 2.3. Statistical analysis Shannon-Wiener and Pielou's diversity index values were calculated based on species cover (Hill, 1973). We used repeated measures ANOVA to investigate the effect of treatment, year and their interaction on vascular plant total cover and species richness. Year was treated as a within group variable; treatment was treated as a between group variable; and plot was treated as a repeated measurement subject. Mauchly's test was used to assess the sphericity assumption. One-way ANOVA was used to investigate the treatment effect on soil ammonium, soil nitrate, total inorganic N and soil pH, with block as random effect. We used Tukey's HSD test for post hoc analysis. We estimated the sensitivity, which was determined as the year when a significant treatment effect became apparent, and the magnitude of the treatment effect. For the first analysis, we treated N dosage as a continuous variable and regressed it against moss cover, vascular plant total cover and the cover of different functional groups. To determine the magnitude of the treatment effect,
experiment (Fig. S2). For moss cover, the response ratio of the low N treatment to the control did not differ from 0, while that of the high N treatment to the control was b0 and showed a decreasing trend over time (Fig. 3a). Deciduous shrubs did not show a response to N addition (Fig. 3b), while the response ratio of evergreen shrubs began to fall below 0 in 2012 and showed a decreasing trend over time under high N treatment (Fig. 3c). The response ratio of forbs remained constant over time (Fig. 3d), while that of graminoid cover began to exceed 0 in 2011 under high N addition and increased until 2013 (Fig. 3e). The cover of the graminoid D. angustifolia started to increase under high N addition in 2013, and the trend continued (Fig. 4a). The cover of the deciduous shrub L. palustre started to decrease in 2014 under high N treatment (Fig. 4b), while the deciduous shrub V. uliginosum showed no obvious trend in response to N addition (Fig. 4c). The cover of the evergreen shrub V. vitis-idaea started to decline under high N relative to the control in 2012, and it became significantly lower under the high N treatment than the control treatment thereafter and almost disappeared from the plot in 2014 (Fig. 4d). 4. Discussion 4.1. The effect of simulated N deposition varied across functional groups In this study, we investigated the effects of simulated N deposition on plant diversity and community structure based on an 8-year N
Treatment we calculated the effect size as LnRR ¼ Ln Cover CoverControl
Statistical analyses were performed with R 3.3.1 (R Core Team, 2016). Diversity indexes were calculated with the Vegan package (Jari Oksanen et al., 2017). We conducted the repeated measures ANOVA with the package EZ (Lawrence, 2016) and performed the multiple comparisons with the Multcomp package (Hothorn et al., 2008). 3. Results 3.1. Effects of N addition across functional types Repeated measures ANOVA indicated no significant effect of N addition on understory species richness (Table 1 and Fig. 1). Furthermore, N addition had no significant effect on vascular plant total cover (Table 1 and Fig. 2a), but it significantly and negatively affected mosscover (Table 1 and Fig. 2b). The relative proportions of the different functional groups of plants in the vascular plant total cover varied over time. Under high N addition, the proportion of evergreen shrubs decreased over time from 66.4% (33.10 ± 11.46) in 2010 to 0.6% (0.2 ± 0.1) in 2017, but even under low N treatment, it decreased from 66.4% (29.93 ± 5.91) in 2010 to 32.4% (10.93 ± 1.07) in 2017, while the proportion remained relatively constant in the control plot. In contrast, the proportion of graminoids increased from 2.7% (1.33 ± 0.54) in 2010 to 59.5% (18.93 ± 11.75) in 2017 under high N addition. The proportions of deciduous shrubs and forbs remained approximately stable. 3.2. Temporal changes in the effects of N addition Diversity under high N treatment showed a decreasing trend (Table 1 and Fig. 1), and richness declined significantly under high N treatment over time (Fig. S1; r2 = 0.423, P b 0.001). Plants sensitivity to N addition varied. Moss cover was significantly and negatively related to N dose in the second year as well as in the subsequent years, and vascular plants total cover showed a decreasing trend with a significant effect observed only in 2014 (Fig. S2). Evergreen shrub cover started to show a negative trend in the second year of the
Fig. 3. Changes in the response ratios of plant cover to N addition during the 8-year study period (a, mosses; b, deciduous shrubs; c, evergreen shrubs; d, forbs; e, graminoids).
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Fig. 4. Effect of N addition on common species over time (a, Deyeuxia angustifolia; b, Ledum palustre; c, Vaccinium uliginosum; and d, Vaccinium vitis-idaea. “*” indicates a significant difference between the high N and control treatments at P b 0.05 as revealed by one way ANOVA in each year).
addition experiment. N addition did not affect species richness, but a decreasing trend was observed under high N addition (Fig. S2), which is consistent with a synthesis of results for boreal forests (Bobbink, 2004). Species richness was not affected but community composition changed (Fig. 3), which was consistent with HRF that plant community under chronic environmental changes may experience a community composition change firstly and finally species loss or immigration (Smith et al., 2009). Our results showed that N addition did not significantly affect vascular plants total cover but significantly decreased moss cover (Table 1 and Fig. 1). Mosses lack a specialized cuticle structure to regulate water or nutrient uptake (Hallbäcken and Zhang, 1998; Makipaa, 1998), and when N deposition is low (e.g. b12 kg N ha−1 yr−1), mosses might take up N at the expense of woody plants (Gundale et al., 2011; Gundale et al., 2014). However, moss N uptake will decrease with increasing N deposition rates (e.g. N12 kg N ha−1 yr−1, Gundale et al., 2014) and moss abundance will decline under high N deposition due to direct NH+ 4 toxicity or competition for light (Nordin et al., 2005; Bret-Harte et al., 2008; Gundale et al., 2014;), which make mosses more vulnerable than vascular plants to increased N deposition (Bobbink et al., 2010; Gundale et al., 2011; Pardo et al., 2011; Gundale et al., 2014). In our current study, N addition significantly increased soil nitrate and total inorganic N (Fig. 5), which might cause a decrease
in moss cover, and this decrease might be partly explained by light competition considering the uneven increase in D. angustifolia (Fig. 4). Vascular plants of different functional groups responded differently to N addition. The relative proportions of deciduous shrubs and forbs were approximately constant (Fig. 3). However, the relative proportion of evergreen shrubs (particularly V. vitis-idaea, Fig. 4d) decreased, while that of graminoids increased (Fig. 3). Deciduous shrubs and graminoids which share common response traits (e.g. generating additional meristems), were good competitors under fertilization; however evergreen shrubs were not (Bret-Harte et al., 2008). Soil inorganic N increased in the organic layer but soil pH did not respond to N addition (Fig. 5). As suggested by the homogeneity hypothesis, this improvement of N availability might have affected interspecific competition and caused a change in community composition (Gilliam et al., 2006, Gilliam et al., 2016). Plants adapted to low N availability, for example, ericaceous dwarf shrubs, investigate their biomass to well-defended leaves, which could slow their growth rate (Cornelissen et al., 2001); as inorganic N availability increased, they failed to compete with fastgrowing graminoids (Cornelissen et al., 2001; Gilliam, 2006; Bobbink et al., 2010). In addition, light often interacts with N fertilization to regulate plant community dynamics (Hautier et al., 2009; Hofmeister et al., 2009; Walter et al., 2016), and this factor might have contributed to the decrease of dwarf shrub V. vitis-idaea. A consistent negative N effect on
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Fig. 5. Effects of N addition on soil chemistry (a, soil ammonium; b, soil nitrate; c, total inorganic nitrogen; and d, soil pH. Bars with the same letter are not significantly different among treatments at P b 0.05).
V. vitis-idaea was found in our study and other fertilization experiments (Strengbom et al., 2001; Manninen et al., 2009), as well as field observations along an N deposition gradient (Strengbom et al., 2003) and this legacy might last long after the termination of N addition (Strengbom et al., 2001). V. vitis-idaea is recognized as a keystone species in boreal forests (Nordin et al., 2005; Hedwall et al., 2013b).This negative N fertilization effect can significantly affect aboveground biomass production and, consequently, the carbon cycle. 4.2. The effect of simulated N deposition changed over long time Understory plants in boreal forests responded rapidly to N fertilization, within one to 3 years (Makipaa, 1998; Strengbom et al., 2002; Du et al., 2014b), partly due to low background N deposition (Gilliam et al., 2016). However, the response of the plant community might exhibit temporal variation with a longer timespan (Nordin et al., 2009; Gilliam et al., 2016). The negative effect of simulated N deposition on mosses and evergreen shrubs accumulated over time, while the positive effect on graminoids increased during the first 4 years but did not change significantly thereafter (Fig. 3). Mosses and evergreen shrubs have been reported to be sensitive and react rapidly to N deposition (Makipaa, 1998; Ostertag and Verville, 2002; Du, 2017). Previous study at our site showed that the biomass of the moss R. rugosum exhibited a decreasing trend after 3 years fertilization (Du et al., 2014b), but this accumulated N deposition might eventually restructure the moss community (Strengbom et al., 2001). A combination of removal and fertilization experiments showed that loss of mosses and evergreen shrubs were compensated by graminoids and deciduous shrubs (Bret-Harte et al., 2008), and a slow recovery of the boreal forest plant community from N fertilization was reported (Strengbom et al., 2001). The most notable changes in vascular plant cover over time under high-level N treatment have been a decrease of the evergreen shrub V. vitis-idaea and increase of the grass D. angustifolia (Fig. 4). D. angustifolia is a mesic nitrophilic plant. A previous study in a tundra
ecosystem found it to be an invasive plant whose abundance increased following N addition, and it tended to be the dominant species in the community (Jin et al., 2016; Zong et al., 2016). The cover of the grass D. angustifolia increased under high-level N, but no increase was observed after 4 years. Similarly, previous studies have shown that the abundance of the grass Deschampsia flexuosa increased linearly with fertilization during the initial 3 years (Strengbom et al., 2002); however, this increase continued for another 3 years and then decreased and remained constant in the following years (Nordin et al., 2009). Variation in growing season weather conditions might interact with N fertilization to affect the plant community (Nordin et al., 2009). For example, a previous study documented that enhanced precipitation affects forbs and grasses differently, and N and precipitation interaction were observed (Zavaleta et al., 2003). This interaction was apparent in 2013 (Fig. 2; Fig. 4), especially for D. angustifolia (Fig. 4a), when extreme growing season precipitation occurred. D. angustifolia cover increased considerably in 2013 under high-level N addition and remained higher thereafter (Fig. 4). However, this N and precipitation interaction was not observed for mosses and V. vitis-idaea, which implies that these plants might hard to recover from negative N fertilization effect. 5. Conclusion Overall, the response of the plant community to 8 years of N addition in an old-growth boreal forest was consistent with the HRF framework and homogeneity hypothesis. N addition did not affect species richness but changed the community composition. Different plant functional groups exhibited varied responses to experimental N addition, and mosses were the most vulnerable. The relative proportion of evergreen shrubs in the vascular cover decreased, while that of graminoids increased under high-level N addition. D. angustifolia cover increased significantly after 4 years, while that of V. vitis-idaea decreased significantly after 3 years and almost disappeared after 5 years. The mechanisms underlying long term community dynamics following increased N deposition remain complex. Future experiments with multiple factors will
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provide valuable evidence to elucidate plants community responses to long term environmental changes. Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.07.350. Acknowledgments This study was supported by the Key Research Program of Frontier Sciences, CAS (QYZDY-SSW-SMC011), National Natural Science Foundation of China (No. 31330012) and the National Key Research and Development Program of China (No. 2017YFC503900). References Bobbink, R., 2004. Plant species richness and the exceedance of empirical nitrogen critical loads: an inventory. Report Landscape Ecology. Utrecht University/RIVM, Bilthoven, The Netherlands. Bobbink, R., Hicks, K., Galloway, J., Spranger, T., Alkemade, R., Ashmore, M., et al., 2010. Global assessment of nitrogen deposition effects on terrestrial plant diversity: a synthesis. Ecol. Appl. 20, 30–59. Bret-Harte, M.S., Mack, M.C., Goldsmith, G.R., Sloan, D.B., Demarco, J., Shaver, G.R., et al., 2008. 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