Plant Soil (2010) 331:377–389 DOI 10.1007/s11104-009-0259-8
REGULAR ARTICLE
Fertilization and litter effects on the functional group biomass, species diversity of plants, microbial biomass, and enzyme activity of two alpine meadow communities Changting Wang & Ruijun Long & Qilan Wang & Wei Liu & Zengchun Jing & Li Zhang
Received: 11 March 2009 / Accepted: 10 December 2009 / Published online: 15 January 2010 # Springer Science+Business Media B.V. 2009
Abstract We conducted a field experiment in two alpine meadows to investigate the short-term effects of nitrogen enrichment and plant litter biomass on plant species richness, the percent cover of functional groups, soil microbial biomass, and enzyme activity in two alpine meadow communities. The addition of nitrogen fertilizer to experimental plots over two growing seasons increased plant production, as indicated by increases in both the living plant biomass and litter biomass in the Kobresia humilis meadow community. In contrast, fertilization had no signifi-
Responsible Editor: Wim van der Putten. C. Wang (*) College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, China e-mail:
[email protected] e-mail:
[email protected] C. Wang : Q. Wang : W. Liu : Z. Jing : L. Zhang Northwest Plateau Institute of Biology, the Chinese Academy of Science, Xining 810008, China R. Long College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730070, People’s Republic of China L. Zhang Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
cant effect on the amounts of living biomass and litter biomass in the K. tibetica meadow. The litter treatment results indicate that litter removal significantly increased the living biomass and decreased the litter biomass in the K. humilis meadow; however, litter-removal and litter-intact treatments had no impact on the amounts of living biomass and litter biomass in the K. tibetica meadow. Litter production depended on the degree of grass cover and was also influenced by nitrogen enrichment. The increase in plant biomass reflects a strong positive effect of nitrogen enrichment and litter removal on grasses in the K. humilis meadow. Neither fertilization nor litter removal had any impact on the grass biomass in the K. tibetica meadow. Sedge biomass was not significantly affected by either nutrient enrichment or litter removal in either alpine meadow community. The plant species richness decreased in the K. humilis meadow following nitrogen addition. In the K. humilis meadow, microbial biomass C increased significantly in response to the nitrogen enrichment and litter removal treatments. Enzyme activities differed depending on the enzyme and the different alpine meadow communities; in general, enzyme activities were higher in the upper soil layers (0– 10 cm and 10–20 cm) than in the lower soil layers (20–40 cm). The amounts of living plant biomass and plant litter biomass in response to the different treatments of the two alpine meadow communities affected the soil microbial biomass C, soil organic C, and soil fertility. These results suggest that the
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original soil conditions, plant community composition, and community productivity are very important in regulating plant community productivity and microbial biomass and activity. Keywords Functional group biomass . Species diversity . Microbial biomass . Enzyme activity . Fertilization . Alpine meadow
Introduction The addition of limiting nutrients to herbaceous plant communities frequently leads to increases in plant production and declines in plant species diversity (Pysek and Leps 1991). The influence of soil fertility on plant primary production, community structure, and diversity has been demonstrated in many plant communities using fertilization experiments (Tilman 1984; Wilson and Tilman 1991; Jonasson 1992). By definition, nutrient-limited systems respond to nutrient additions with increases in primary production, but long-term fertilization studies suggest that changes in species composition following fertilization may be more long-lasting than changes in production (Tilman 1984, 1987). A number of prominent theories have focused on resource competition and its potential role in determining the effects of productivity on species diversity in plant communities (Huston and DeAngelis 1994). Most of these theories suggest that competition for light occurring among living plants at high productivity results in greater competitive exclusion and reduced species richness (Abrams 1995). Although resource competition among living plants is undoubtedly important in limiting the diversity of many plant communities, but generally do not consider the potential role of accumulated plant litter in contributing to reduced diversity at high levels of plant productivity. Plant litter can play an important role in structuring plant communities by directly and indirectly affecting individuals and populations (Facelli and Pickett 1991). The inhibition of plant diversity by litter is most commonly observed in highly productive, undisturbed environments, in which litter accumulation can be quite high (Tilman 1993). Litter production is dependent on the vegetation cover (an exponential relationship) and is also influenced by soil fertility (Descheemaeker et al. 2006). Together,
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these studies suggest that the amount of litter may influence species diversity along fertility and productivity gradients. Plants alter soil processes in many ways, ranging from differences in detrital inputs and nutrient use, to alterations to microclimate or disturbance cycles (Hobbie 1992). Many of the most important biogeochemical processes influenced by plants occur in the soil, yet belowground processes remain the least understood component of ecosystems (Sugden et al. 2004). Plant species identity also affects ecosystem processes (Hobbie 1992; Vinton and Burke 1997) such as nutrient cycling (Wedin and Tilman 1990), microbial biomass and composition (Bardgett et al. 1999), and soil enzyme activity (Kourtev et al. 2002). However, no single study has directly compared the responses of vegetation cover, functional groups biomass, species diversity, microbial biomass, and enzyme activity to differences in plant litter biomass and fertilization inputs between two alpine meadow communities that differ primarily in their levels of soil fertility, plant composition, and plant community biomass. The Tibetan plateau is the highest plateau in the world and is known as “the third pole” of the world. One of the typical types of zonal plateau vegetation on the Qinghai-Tibetan plateau is Kobresia, which dominates alpine meadows (China vegetation edit commission 1980). This meadow is found at altitudes from 2,000 m to 5,000 m, which have a cold, semihumid climate characterized by a long, cold dry season and a short, warm growing season of only 90–150 days. These climatic attributes result in low primary productivity in alpine Kobresia meadows (Wang et al. 1995). A previous study (Bowman et al. 1993) demonstrated that alpine primary production is nutrient limited, and that this limitation is greater in dry meadows than in wet meadows. Thus, to understand the effects of litter in contributing to changes in two alpine meadows community structure, species diversity, microbial biomass, and enzyme activity, it is necessary to manipulate litter levels in concert with fertilization. The goals of our study were: (1) to examine effects of nitrogen enrichment on plant litter biomass and living plant biomass, vegetation cover, and species diversity within two alpine meadows; (2) to examine the effects of litter in contributing to changes in two alpine meadows community structure, species diversity, microbial biomass, and enzyme activity that are
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associated with nitrogen enrichment and increased productivity. Three hypotheses were addressed: (1) both living plants and plant litter would stimulate microbial biomass and activity, and the enhancement of microbial biomass and activity would occur in treatments containing both live plants and added litter; (2) within each community, plant functional groups will respond differently to nutrient additions; and (3) the species diversity in both communities will be affected by fertilization, but the responses will differ between the two communities. In this study, we report the results of a 3-year field experiment in which the experimental addition of nitrogen fertilizer was accompanied by the experimental removal of plant litter in two alpine meadows communities.
Materials and methods Field site This study was conducted at Haibei Research Station, the Chinese Academy of Sciences (37°32′N, 101°15′ E, altitude 3240 ma.s.l.). The average annual precipitation recorded at the station from 1976–2001 was 560 mm, with 85% of that rainfall occurring during the growing season from May to September. The average annual air temperature from 1976–2001 was −1.7°C. The experimental site was located within a typical alpine meadow. The species richness in this type of vegetation is high, with 25–40 species per 1 m2, among which mesophilus species are predominant. The dominant species are Kobresia humilis, K. capillifolia, and Carex atrifusca, with many accompanying species such as Poa polygonum and Festuca modesta. The grass community typically has one to two layers, with a height of 45–60 cm for the highest grasses and an overall ground cover of 60–95%. Alpine swamp meadows are dominated by hydrophytes and mesohydrophytes, such as Kobresia tibetica, Blysmus sinacompressus, and Carex scabriostris. The accompanying species include Carex atrifusca, Saussurea stella, etc. The grass community usually possesses one layer, with an average height of 10–25 cm and an overall ground cover of 80– 96%. The soils at the study site are classified as swamp meadow soil and alpine meadow soil (Zhou 2001).
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Experimental design and setup In mid-April 2005, we established twelve 4 m×3 m experimental blocks at each of the study sites (a typical alpine meadow and an alpine swamp meadow). The blocks were arranged in two parallel columns of six blocks each that were separated by a 2-m buffer strip. Blocks within each column were separated by buffer zones of 1 m. Within each block, four 1×1 m treatment plots were established and placed so that there was a 0.5 m buffer between each plot. Two levels of nitrogen fertilization (0 g/m2 and 20 g/m2) and two levels of litter manipulation (litter removed and litter left intact) were applied to 1×1 m plots in a factorial design. Thus, there were four treatment combinations for each of the study sites: nitrogen added (+N) with litter removed (LR); nitrogen added (+N) with litter left intact (LI); litter left intact (LI) with no nitrogen (-N); litter removed (LR) with no nitrogen (-N). Litter was removed from the appropriate plots in November 2005, 2006, and 2007 by first clipping around the perimeter of the plots. The litter was subsequently well shaken by hand to reduce the seed content, and the litter mat was lifted gently by hand in order to remove it. The litter was removed in November to eliminate the current year’s production that had fallen into the plots as litter. In March 2006 and 2007, the small amount of litter that had blown into these plots over the winter was removed by hand. The fertilization treatment consisted of the annual application of 0 g/m2 or 20 g/m2 of a commercial CO (NH2)2 fertilizer in pellet form (46.65% N) that was applied in late May of 2005, 2006, and 2007. Sample collection and processing Vegetation sampling In late August 2005–2007, the aboveground living plant biomass and litter were harvested from two 25 cm×25 cm sampling quadrats within each 1 m× 1 m plot. Sampling was conducted at the time of peak biomass for each community. Peak biomass occurred in the two alpine meadows in approximately late August of each year. Each of these quadrats was centered within a plot. The quadrats were selected randomly and located at least 50 cm from the side of the plots to avoid edge effects. The total ground cover
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of the vegetation was measured for all plots. For each plot, the height and percentage of the cover of each plant species was recorded, and the vegetation was clipped off flush with the ground in each quadrat. The frequency of each plant species was calculated for each plot. The harvested plants were separated into four functional groups (grasses, sedges, legumes, and forbs). The dominant species was selected according to its relative percentage of cover for the plot. Root biomass was sampled in ten soil cores (diameter of 5 cm) per plot at a depth of 0–40 cm. The soil cores were crumbled by hand, passed through a 2-mm mesh screen to remove roots, and then stored at 4°C until analysis. The root biomass sieved from the core was combined with the roots removed from the remainder of the sample to determine the total root biomass. All vegetative material was dried (48 h at 70°C) and weighed. Seed germination was counted at the end of April in 2005–2007 in each quadrat. Microbial biomass carbon and soil enzyme activity Microbial biomass carbon (biomass C) was determined by the fumigation-extraction (FE) method (Vance et al. 1987). Three subsamples of moist soil (equivalent to 5.0 g dry soil) were extracted with 20 ml 0.5 M K2SO4. The samples were shaken for 30 min, filtered, and frozen at −20°C. Simultaneously, three other subsamples of soil (also equivalent to 5.0 g of dry soil) were fumigated with ethanol-free chloroform for 24 h at 2°C, and then extracted and frozen. Biomass C (BC) was calculated from BC =2.22 EC, where EC is equal to (C extracted from fumigated soil)—(C extracted from nonfumigated soil). The concentration of extracted carbon was determined by an automated TOC Analyzer (Shimadzu, TOC5000A, Japan). The soil enzyme activities of urease, protease, alkaline phosphatase, and invertase were analyzed using the methods described in the soil enzyme analysis manual (Guan 1986). Soil sampling Soils from each plot were sampled by combining six soil cores (5 cm in diameter) obtained in a V-shaped pattern and splitting them into 0–10 cm, 10–20 cm, and 20–40 cm sections. Samples were combined based on plot and depth in the field, cooled to 4°C, returned to the laboratory, and processed within
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2 days. Samples were collected on 20 August 2005, 2006, and 2007. Soil moisture was measured gravimetrically after drying at 105°C for 24 h. Calculations and statistical analyses Species importance values were calculated for the herb layer. Two indices were selected to estimate diversity according to Pielou (1969). The first index is plant species richness (S), represented by the number of species recorded in each plot (we present species richness for each plot as the sum of species found across two community sampling quadrats). The second, the Shannon-Wiener’s index of diversity, s P is H 0 ¼ Pi LnPi , where Pi stand for relatively i¼1
important value of the species i in each plot (Pi = (relative cover + relative height + relative frequency)/3). For vegetation samples, the means obtained for the quadrats in each plot were used to calculate the treatment means. Treatment effects on living plants, plant litter biomass, Shannon-Wiener index, abundance of functional groups, microbial biomass C, and soil enzyme activity were examined using a randomized block 2 × 2 factorial ANOVA. This allowed us to test for the main effects of nitrogen and litter and to evaluate their potential interaction. Tests for significant differences among treatments were conducted by analysis of variance (ANOVA) with least significant difference (LSD) tests. Analyses were conducted using SPSS 10.0 software (Putian Electron Technology).
Results Response of living biomass and litter biomass to treatments In the K. humilis meadow, nitrogen enrichment increased plant community productivity, as reflected by significant increases in both the living biomass and litter biomass (Fig. 1) in response to fertilization. In contrast, N fertilization had no significant effect on the living biomass or litter biomass (Fig. 1) in the K. tibetica meadow. Similarly, a separate analysis comparing the biomass responses in the litter removal (LR) and litter intact (LI) treatments indicated that, in the K. humilis meadow, litter removal significantly
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N: F1, 11 = 1.73, P = 0.218 N: F1, 11 = 320.88, P 600
Living biomass (g/m2)
Fig. 1 Treatment effects on living biomass and litter biomass in the K. humilis and K. tibetica meadows. Bars represent means + SD. Significant differences obtained by 2×2 factorial ANOVA are presented. Biomass values with the same letters are not significantly different (P>0.05) in the same community between the different treatment levels
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0.001
a
510
L:F1, 11 = 270.55, P 0.001
500
b
400
b
480
c
200
470
100
460 +N+LR
+N+LI
-N+LR
L: F1, 11 = 0.79, P = 0.383 N×L: F1, 11 = 0.012, P = 0.915 a a a
500 490
300
0
a
450
-N+LI
+N+LR
+N+LI
-N+LR
-N+LI
N: F1, 11 = 0.01, P = 0.919
Litter biomass (g/m2)
90
a
80
N: F1, 11 = 92.93, P
0.001
L: F1, 11 = 65.72, P
0.001
L: F1, 11 = 0.45, P = 0.508 64 62
70
b
60
60
N×L: F1, 11 = 2.22, P = 0.152 a a a
58
50 40
c
56
c
30
54
20
52
10
50
0
a
+N+LR
+N+LI
-N+LR
K. humilis meadows
increased the living biomass and decreased the litter biomass (Fig. 1). However, in the K. tibetica meadow, there was no effect of litter manipulation on either living or litter biomass (Fig. 1).
-N+LI
48
+N+LR
+N+LI
-N+LR
-N+LI
K. tibetica meadows
(Fig. 2). However, fertilization and litter removal had no impact on forb biomass in the K. tibetica meadow (Fig. 2). Sedge biomass was not significantly affected by nutrient enrichment or litter manipulations in either alpine meadow community (Fig. 2).
Functional group biomass in response to treatments In the K. humulis meadow, the increase in plant biomass that was observed in response to nitrogen enrichment and litter removal reflects a strong positive effect of fertilization on grass biomass (Fig. 2). In contrast, neither fertilization nor litter removal had any impact on grass biomass in the K. tibetica meadow (Fig. 2). Both the addition of nitrogen and leaving the litter intact led to a significant decrease in legume biomass, whereas litter removal and no addition of nitrogen led to a significant increase in legume biomass in the humilis meadow (Fig. 2). There was no legume functional group in the K. tibetica meadow. The biomass of forbs, which was much lower than that of grasses overall, was significantly affected by nitrogen addition and litter removal in the K. humilis meadow
Changes in the cover of functional groups in response to the different treatments The percentage of grass cover increased significantly in response to nitrogen enrichment, whereas the cover of legumes and forbs were reduced significantly by nitrogen addition in the K. humilis meadow. However, nitrogen enrichment had no impact on sedge cover in the K. tibetica meadow (Table 1). Similarly, a separate analysis comparing the responses of functional groups to the litter manipulation indicates that litter removal drastically increased the cover of functional groups, but had no effect on the cover of sedges in the K. humilis meadow. In addition, the nitrogen enrichment and the litter removal treatments did not significantly influence the percent cover of functional groups in the K. tibetica meadow (Table 1).
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N: F1, 11 = 298.03, P 0.001 Grass biomass (g/m2)
a
L: F1, 11 = 255.16, P 0.001 a
b
N: F1, 11 = 0.005, P = 0.928 L: F1, 11 = 0.31, P = 0.589 N L: F1, 11 = 0.004, P = 0.952 a a a
b c
Legume biomass (g/m2)
N: F1, 11 = 5.71, P = 0.03 a
L: F1, 11 = 1740.95, P 0.001 b
c
c
N: F1, 11 = 5.88, P = 0.249
N: F1, 11 = 3.88, P = 0.05
L: F1, 11 = 0.50, P = 0.608
L: F1, 11 =111.65, P 0.001
Forb biomass (g/m2)
a a
b c
N L:aF1, 11 = 0.76, P = 0.393 a a
c
N: F1, 11 = 0.81, P = 0.378 L: F1, 11 = 0.66, P = 0.428
Sedge biomass (g/m2)
Fig. 2 Treatment effects on grass biomass, legume biomass, forb biomass, and sedge biomass in the K. humilis and K. tibetica meadows. Bars represent means + SD. Significant differences determined by 2×2 factorial ANOVA are presented. Biomass values with the same letters are not significantly different (P>0.05) in the same community between the different treatment levels
Plant Soil (2010) 331:377–389
N L: F1, 11 =2.32, P = 0.143 a a
N: F1, 11 = 90.49, P = 0.067 L: F1, 11 = 0.10, P = 0.807 a
N L: F1, 11 = 0.13, P = 0.726 a
a a
K. humilis meadows
a
K. tibetica meadows
a
Plant Soil (2010) 331:377–389 Table 1 ANOVA results for comparisons of the mean percent cover of functional groups among experimental treatments in two alpine meadow communities
383 Source
SS (Total) df
K. humilis meadow Nutrient treatment Grasses
2324.08
Sedges
33.00
MS
F
P
1,11 2324.08 131.43 0.0001 1,11
1.33
0.42 0.5310
Legumes
2148.92
1,11 2106.75 499.63 0.0001
Forbs
2285.66
1,11 2133.33 140.04 0.0001
K. tibetica meadow Nutrient treatment Grasses
6.73
1,11
1.02
1.79 0.2108
Sedges
20.25
1,11
0.08
0.04 0.8430
Forbs
14.92
1,11
0.75
0.53 0.4835
K. humilis meadow Litter treatment
K. tibetica meadow Litter treatment
Grasses
1432.48
Sedges
10.00
1,11 1331.41 131.74 0.0001 1,11
0.33
0.35 0.5701
Legumes
1181.66
1,11 1140.75 278.79 0.0001
Forbs
2192.98
1,11 2162.76 715.75 0.0001
Grasses
2.06
1,11
0.02
0.12 0.7560
Sedges
11.66
1,11
0.33
0.29 0.5995
Forbs
26.23
1,11
1.02
0.41 0.5388
according to the community type. In the K. humilis meadow, species diversity decreased significantly with nitrogen enrichment and when the litter was left intact (Fig. 4). In contrast, the species diversity of the K. humilis meadow increased in response to litter removal when no N was added. No effects on plant species richness or diversity were observed in the K. tibetica meadow in response to the fertilization and litter treatments carried out in 2005–2007 (Fig. 4).
Species richness in response to the different treatments Both fertilization with nitrogen and leaving the litter intact led to significant declines in species richness in the K. humilis meadow (Fig. 3). Similarly to the biomass responses, a separate analysis showed that the nitrogen enrichment and litter removal treatments had no impact on species richness in the K. tibetica meadow (Fig. 3).
Microbial biomass C Species diversity responses to different treatments The results for the response of microbial biomass C to nitrogen enrichment and litter removal treatment were similar to those of species diversity on the community
The response of species diversity to the nitrogen enrichment and litter removal treatments differed
N: F1, 11 = 5.86, P = 0.04
N: F1, 11 = 2.77, P = 0.112 L: F1, 11 = 2.77, P = 0.112
L: F1, 11 =88.82, P 0.001 a
Species richness
Fig. 3 Treatment effects on species richness in the K. humilis and K. tibetica meadows. Bars represent means + SD. Significant differences obtained by 2×2 factorial ANOVA are presented. Species richness values with the same letters are not significantly different (P>0.05) in the same community between the different treatment levels
N L: F1, 11 = 0.06, P=0.962 a
a b
K. humilis meadows
b
a a
K. tibetica meadows
a
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Plant Soil (2010) 331:377–389 N: F1, 11 =0.05, P = 0.982
N: F1, 11 = 169.90, P = 0.048
L: F1, 11 = 0.47, P = 0.499 L: F1, 11 =281.07, P 0.001 N L: F1, 11 = 0.036, P = 0.852
a
a
a
a
Spicies diversity (H')
Fig. 4 Treatment effects on species diversity (H′) in the K. humilis and K. tibetica meadows. Bars represent means + SD. Significant differences obtained by 2×2 factorial ANOVA are presented. Species diversity values with the same letters are not significantly different (P>0.05) in the same community between the different treatment levels
a
K. humilis meadows
type. In the K. humilis meadow, microbial biomass C increased significantly in the different soil layers in response to the nitrogen enrichment and litter removal treatments (Table 2). In contrast, the K. tibetica meadow microbial biomass C was not altered in response to either nitrogen enrichment or litter removal treatments (Table 2). Enzyme activity Significant differences (P
