Soil Biology & Biochemistry 42 (2010) 1588e1595

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Soil Biology & Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

Tree species-mediated spatial patchiness of the composition of microbial community and physicochemical properties in the topsoils of a tropical montane forest
Masayuki Ushio a, *, Kanehiro Kitayama a, b, Teri C. Balser c
a b c

Center for Ecological Research, Kyoto University, 2-509-3 Hirano, Otsu, Shiga 520-2113, Japan Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake, Sakyo-ku, Kyoto 606-8502, Japan Department of Soil Science, University of Wisconsin e Madison, Madison, WI 53706, USA

a r t i c l e i n f o
Article history: Received 30 September 2009 Received in revised form 30 May 2010 Accepted 31 May 2010 Available online 11 June 2010 Keywords: Condensed tannins Conifer (Podocarpaceae) Enzyme activity Lipid prole Plantesoil feedback Soil microbial community Tropical montane forest

a b s t r a c t
To evaluate the importance of plantesoil feedbacks in forest ecosystems, it is fundamental to understand the spatial range within which plant species control soil physicochemical and microbial properties. We investigated the spatial pattern of soil properties associated with canopy trees in a tropical montane forest on Mt. Kianbalu, Borneo. We analyzed soil physicochemical properties and microbial communities (biomarker lipid abundance) as a function of soil depth and distance from the tree trunk of a conifer (Dacrydium gracilis) or a broadleaf tree (Lithocarpus clementianus). The concentration of condensed tannins and fungi-to-bacteria were higher beneath Dacrydium than beneath Lithocarpus. Furthermore, carbon-degrading enzyme activities were lower beneath Dacrydium. These effects of the tree species were more distinct on soil properties beneath the tree crown than on those outside the tree crown. These effects appeared to be largely due to differences in litter chemistry, and the distinct set of soil properties formed corresponding to the above canopy crown. In conclusion, the species-rich forest on the tropical mountain contains spatially distinct units of soil properties associated with canopy trees, and this spatial pattern can inuence ecosystem dynamics in the forest through plantesoil feedback effects. 2010 Elsevier Ltd. All rights reserved.

1. Introduction Feedbacks between plants and soil are a potential driver of plant community and ecosystem dynamics (Wardle et al., 2004). It has been suggested that plant species can control soil physicochemical properties such as soil pH, moisture and C-to-N ratio (Binkley, 1995), microbial communities, and enzyme activities through speciesspecic litter chemistry (Ushio et al., 2008; Strickland et al., 2009). Plant-mediated changes in soil properties subsequently change soil nutrient availability, which in turn feeds back to affect plant community dynamics (Frelich et al., 1993; Wardle, 2002). Empirical and theoretical studies indicate that such litter-mediated plantesoil feedbacks can regulate nutrient cycling in an ecosystem (Hobbie, 1992; Vitousek, 2006) and can drive plant community dynamics (Miki and Kondoh, 2002; Binkley and Menyailo, 2005). Soil microbial community and physicochemical properties can be directly controlled by plant species-specic litter chemistry

* Corresponding author. Tel.: 81 77 549 8018; fax: 81 77 549 8201. E-mail address: ong8181@gmail.com (M. Ushio). 0038-0717/$ e see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.05.035

(Binkley, 1995; Strickland et al., 2009). In a forest ecosystem, a distinct spatial pattern of soil properties associated with plant canopies can be formed corresponding to the size of inuential canopies because plant species produce a unique set of foliar chemicals (e.g., various phenolic compounds) and because the foliar chemicals affect soil microbial and chemical properties (Kuiters, 1990; Kraus et al., 2003). Since different tree species produce leaf litter with different chemical composition, this spatial pattern of soil properties associated with plant canopy can be more distinct in a species-rich forest (i.e., various tree species in a spatially narrow range) than in a species-poor forest (e.g., a single species in a spatially narrow range) such as Hawaiian forest ecosystem (Crews et al., 1995). Both microbial and physicochemical properties can jointly inuence soil enzyme activity (Balser and Firestone, 2005; Allison et al., 2007; Sinsabaugh et al., 2008), and the soil enzyme activity is a regulator of soil nutrient availability. Therefore, the pattern of soil properties associated with plant canopy can inuence the consequence of plantesoil feedbacks because soil nutrient availability can be one of the primary factors that inuence subsequent plant growth and community dynamics (Binkley and Menyailo, 2005; Vitousek, 2006).

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Variation in leaf chemistry occurs among tree species, and decomposition rate is inherently slow due to cool climate and the low concentrations of available nutrient in the tropical montane forest on Mt. Kinabalu, Borneo. These conditions will likely induce a distinct spatial pattern of soil properties associated with plant canopy. The forest is composed of diverse evergreen broadleaf trees species interspersed with conifers (Aiba et al., 2002). The leaf chemistry of the conifers is different from that of the broadleaf trees, especially in the concentration of condensed tannins (Suzuki, S. unpublished data). These differences may cause differences of soil physicochemical properties, microbial communities and enzyme activities beneath the tree species since condensed tannins have strong inuences on both soil physicochemical and microbial properties (Scalbert, 1991; Kraus et al., 2003). In addition, the surface organic layer is thicker and the retention time of litter is longer due to the relatively slow decomposition caused by the lower air temperatures compared to that at lowland sites (Kitayama et al., 2000). Therefore, above-ground litter can exert prolonged inuences on soil physicochemical and microbial properties although below-ground litter and live roots may also exert similar inuences on soil processes (e.g., Von Arnold et al., 2005) and microbial communities (e.g., Lejon et al., 2005). To evaluate the potential importance of plantesoil feedbacks in forest ecosystems, it is fundamental to understand the spatial range within which plant species control soil physicochemical and microbial properties, as well as the strength of plant controls over soil properties (Wardle, 2002). The spatial range of plant control on soil properties potentially determines the extent of nutrientavailability gradient around the plant, while the strength of inuence directly determines the subsequent plant performance (e.g., productivity, growth rate and biomass) on the soil. Recent studies have examined the strength of the plant control on soil physicochemical and microbial properties, and/or subsequent feedbacks on the plant performance (Porazinska et al., 2003; Bartelt-Ryser et al., 2005; Kardol et al., 2007; Liang et al., 2007; Kulmatiski et al., 2008; Ushio et al., 2008), but few studies have addressed the spatial range of plant control of soil properties (Saetre, 1999; Saetre and Bth, 2000), which determines the spatial scale of the plantesoil feedbacks. In this study, our primary objective was to quantify the spatial pattern of soil properties associated with plant canopy. Since litter chemistry can inuence soil physicochemical and microbial properties, we hypothesized that: (1) the spatial pattern of soil properties would correspond with the extent of the canopy crown (i.e., litter fall pattern of the tree), (2) the soil physicochemical and microbial properties of surface organic soil would show more distinct spatial patterns than those of deeper mineral soil. As our secondary objective, we briey explore the relationships among soil physicochemical properties, microbial community and the enzyme activities. 2. Materials and methods 2.1. Research site This study was conducted within a permanent plot in a primary montane forest on the south slope of Mt. Kinabalu in Sabah, Borneo, Malaysia (summit height, 4095 m above sea level; 6 050 N, 116 330 E). The research plot is at 1560 m above sea level, near the Park Headquarters of the Kinabalu Park. The climate is humid and tropical with mean annual air temperature of 18  C, and mean annual precipitation of 2714 mm (Aiba and Kitayama, 1999). This area does not have marked seasonality but does vary slightly in monthly precipitation (Brief data of precipitation can be obtained from a website of the Sabah Forest Department: http://www.forest.sabah.gov.my/caims/).

The plot is covered with evergreen broadleaf trees interspersed with conifers (the relative basal area of conifers is ca. 15%). There are 109 tree species per hectare with >10 cm diameter at breast height in the plot (Aiba et al., 2002). Because of the relatively low temperature and modest litter supply rate, the decomposition rate of standing litter is low compared to that at lowland sites and therefore standing litter in this plot is thick. The plot is in the last stage of pedogenesis and the soil contains a low concentration of available phosphorus (e.g., a negligible concentration of inorganic phosphorus in soil: 37.04 mg g1 of NaHCO3 extractable inorganic phosphorus, 34.49 mg g1 of NaOH extractable inorganic phosphorus and 12.85 mg g1 of diluted HCl extractable inorganic phosphorus), which is thought to limit plant growth (Kitayama et al., 2000, 2004). 2.2. Soil sampling We selected four replicate trees from two dominant tree species in the plot: one was a conifer species, Dacrydium gracilis (Podocarpaceae); and the other was a broadleaf species, Lithocarpus clementianus (Fagaceae). Leaves of Dacrydium contain a higher concentration of condensed tannins than those of Lithocarpus (Dacrydium, 6.38%; Lithocarpus, 0%; Suzuki S, unpublished data). We sampled soils on one transect beneath each of the replicates in August 2007. Soils were sampled from four soil layers (upper organic layer, O1 [soil depth, ca. 0e3 cm]; lower organic layer, O2 [ca. 3e6 cm]; upper organic mineral layer, A1 [ca. 6e10 cm]; lower organic mineral layer, A2 [ca. 10e15 cm]), and from three distances from a tree trunk (INSIDE, DRIPLINE, and OUTSIDE regions of a tree crown [ca. 3, 6, and 12 m apart from a tree trunk, respectively]). Both the soil depths and the distances from a tree trunk vary from place to place, depending on the thickness of each soil layer and the horizontal radius of the tree crown, respectively. In addition, standing litter and fresh litter at the three positions (INSIDE, DRIPLINE and OUTSIDE) were collected for analysis of organic C, total N, and phenolic content (i.e., total phenolics and condensed tannins). Fresh litter was collected by placing 60 cm 90 cm litter traps on the forest oor for two weeks. In total, we collected 96 soil samples (2 tree species 4 soil layers 3 distances 4 replicates) and 24 litter samples (2 tree species 2 litter layers 3 distances 4 replicates). Soil samples were taken to the laboratory immediately after collection, and roots removed by hand. The samples were refrigerated at 4  C for up to 10 days, and analyzed for moisture, pH in water and pH in 0.01 N KCl, organic C and total N, and the concentration of total phenolics and condensed tannins. Organic C and total N contents were measured using a macro-corder (JM 1000CN, J-SCIENCE LAB Co, Ltd, Japan). For total phenolics and condensed tannins measurements, 50% MeOH extracts of dried soil or litter samples were analyzed. The concentration of total phenolics was determined by the FolineCiocalteau method using a standard curve prepared using tannic acid (Waterman and Mole, 1994). The concentration of condensed tannins was determined by the acidebutanol method using a standard curve prepared using cyanidinchloride (Porter et al., 1986). 2.3. Microbial-lipid biomarkers Immediately after soil sampling a subset of soil samples collected was homogenized, frozen, and then freeze-dried for lipid analysis. We used microbial-lipid analysis (extraction of signature lipid biomarkers from the cell membrane and wall of microorganisms [White and Ringelberg, 1998]) to assess the microbial community composition of each freeze-dried soil sample. We extracted, puried and identied PLFAs from microbial cell membranes using a hybrid lipid extraction based on a modied

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Bligh and Dyer (1959) technique, combined with fatty acid methyl ester analysis (FAME) as described by Microbail ID Inc. (Hayward, CA) (see Kao-Knifn and Balser, 2007). All glassware was baked at 475  C for 4 h. Lipids were extracted from about 1 g of freeze-dried soil using chloroform-methanol extraction with phosphate buffer (potassium phosphate [3.6 ml], methanol [8 ml], and CHCl3 [4 ml]), shaken for 1 h and centrifuged. The supernatant was then decanted into 30-ml tubes and potassium phosphate buffer and chloroform were re-added and the tubes were vortexed for 1 min. The phases were allowed to separate overnight at room temperature. The top layer was aspirated off, saving the chloroform phase, and the volume was reduced in a RapidVap. We then followed the procedure for FAME as given by Microbial ID Inc; sodium hydroxide was added for saponication and the solution was heated in a water bath for 30 min, followed by mild alkaline methanolysis. Fatty acids were analyzed using a HewlettePackard 6890 Gas Chromatograph equipped with a ame ionization detector and split/ splitless inlet and a 25 m 0.2 mm inside diameter 0.33 mm lm thickness Ultra 2 (5%-phenyl, 95% methyl) capillary column (Agilent) using hydrogen as the carrier gas, N2 as the make-up gas, and air to support the ame. Gas chromatograph conditions were set by the MIDI Sherlock program (MIDI, Inc. Newark, DE). Peaks were identied by using bacterial fatty acid standards and Sherlock peak identication software (MIDI, Inc. Newark, DE). Fatty acids were quantied by comparison of peak areas from the sample compared with peak areas of two internal standards, 9:0 (nonanoic methyl ester) and 19:0 (nonadeconoioc methyl ester), of known concentration. From there we calculated abundance of total lipid and each indicator lipid. Total lipid abundance was calculated as a sum of lipids of which chain length was from 10 to 20. Indicator lipids used for calculation were as follows. Total bacterial biomass was estimated by the sum of the abundance of i14:0, 15:0, i15:0, a15:0, i16:0, i17:0, a17:0, 16:1u7, cy17:0 and cy19:0 (Frostegrd et al., 1993). Gram-positive bacteria were represented by the branched lipids including i14:0, i15:0, a15:0, i16:0, i17:0, and a17:0 (Zelles et al., 1995), whereas Gram-negative bacteria were represented by 16:1u7, cy17:0 and cy19:0 (Ratledge and Wilkinson, 1988; Yao et al., 2000). Fungal biomass was estimated by the abundance of 18:2u6,9 (Frostegrd et al., 1993), and indicators of actinomycetes were 16:0 10Me and 17:0 10Me (White et al., 1979; Zelles et al., 1995). Indicators of arbuscular mycorrhizal fungi (AMF) and protozoa were 16:1u5 (Olsson et al., 1995) and 18:3u6,9,12, respectively. Fungi-to-bacteria ratio and Gram-positive bacteria to Gram-negative ratio were calculated as 18:2u6,9/(sum of all bacterial lipid), and (sum of the branched lipids)/(sum of the monounsaturated and cyclopropyl lipids), respectively. Complete details of lipid abundance in each soil layer are presented in Supplementary Data (Table S2e5). 2.4. Enzyme activities We measured the activities of acid phosphatase, b-D-glucosidase, phenol oxidase, and peroxidase. Soil samples were kept in a refrigerator at 4  C for up to 10 days before the enzyme assay. First, 5 g of wet soil per sample was mixed with 50 ml of 50 mM acetate buffer (pH 5.0), and shaken vigorously for 30 min. For acid phosphatase and b-D-glucosidase analysis, the modied method of Tabatabai and Bremner (1969) was used. Briey, 500 ml of soil suspension was added to a 1.5-ml microtube, and 500 ml of 5 mM substrate solution was added to each microtube. Substrates were p-nitrophenyl phosphate for acid phosphatase and p-nitrophenyl b-D-glucopyranoside for b-D-glucosidase. The mixture was incubated for 2 h at 20  C. Following the incubation, tubes were centrifuged at 10,000 rev min1 for 5 min and a 0.5-ml aliquot of clear supernatant was taken from each tube and transferred to

a 6-ml glass vial containing 0.1 ml of 1 N NaOH in order to stop the reaction and develop the color. The solution was brought to a volume of 5 ml using deionized water. The assay mixtures were then shaken and absorbance was measured at 410 nm. A standard curve was developed using p-nitrophenol. For phenol oxidase and peroxidase, 3,4-dihydroxy-L-phenylalanine (DOPA) was used as substrate. For phenol oxidase, a total of 1 ml of soil suspension was added to 1 ml of 5 mM DOPA solution (0.1 ml of 0.3% H2O2 was further added only for peroxidase). The mixture was incubated for 1 h at 20  C. Following incubation, tubes were centrifuged at 10,000 rev min1 for 5 min and 1.5 ml of the supernatant was transferred to a new 6-ml vial. The solution was brought to a volume of 3 ml using deionized water. The absorbance of samples was measured at 460 nm. A standard curve was developed using DOPA and tryrosinase. 2.5. Statistics We used a general liner mixed model (GLMM) to test the effects of tree species and soil layer on soil properties, lipid abundance, and soil enzyme activities (Model formula: factor e tree species soil layer tree species soil layer), including tree replicate as a random factor (by using nlme package of R; Pinheiro et al., 2009). The effect of distance from a tree trunk was considered by performing the same GLMM separately for each distance. Values were log-transformed to improve normality before GLMM. To estimate microbial community ngerprint, principal component analysis (PCA) was performed for the relative proportion of identied microbial-lipid abundance (i.e., mol percent). Those lipids which were lower than 0.5% in relative abundance were excluded from the PCA. To ensure normality in our lipid data set, we transformed values by taking the negative arcsine of the square root of the mole percent (Waldrop et al., 2000; Balser and Firestone, 2005). The compositional dissimilarity of microbial communities was assessed by calculating the Euclidean distances between the values of principal components (PCs) of Dacrydium and those of Lithocarpus in each treatment, i.e., square root of ([PC1 Dacrydium e PC1 Lithocarpus]2[PC2 Dacrydium e PC2 Lithocarpus]2). Euclidean distances were calculated on all combinations of Dacrydium and Lithocarpus samples in the same treatment. The signicance of the difference in the Euclidean distances was tested by permutation test (999 permutations). In order to identify factors explaining soil enzyme activities, we conducted redundancy analysis (RDA; Rao, 1964) with soil enzyme activities as a dependent variable and with selected soil physicochemical properties (i.e., pH (H2O), organic C, total phenolics, condensed tannins) and lipid principle components (PC1 and PC2) as explanatory variables (by using vegan pakcage of R; Oksanen et al., 2008). Some of functionally correlative variables (i.e., pH (KCl) from pH (H2O), and total N from organic C) were excluded from the RDA. RDA was applied separately to O and A layer because the concentrations of condensed tannins were not detectable in most A layer samples. The proportion of explained variation was calculated by using adjusted R-squared values as described by Peres-Neto et al. (2006). All variables were standardized before the analysis. The signicance of each explanatory variable was tested by a permutation test (999 permutations). All statistical analyses were performed in the statistical environment R (R Development Core Team., 2009). 3. Results 3.1. The spatial pattern of soil physicochemical properties and litter chemistry The concentration of condensed tannins and soil pH showed a distinct spatial pattern depending on tree species, soil layer, and

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distance from a tree trunk (Table 1 and S1). Fresh litter collected from the INSIDE region beneath Dacrydium and beneath Lithocarpus consisted of ca. 50% Dacrydium leaves and ca. 40% Lithocarpus leaves, respectively (dry weight base; data not shown). The concentration of condensed tannins in surface soils was much higher beneath Dacrydium than beneath Lithocapus (Table S1). The soil pH of O1 layers was lower at the INSIDE region of Dacrydium than at the INSIDE region of Lithocaprus (Table S1). In contrast, deeper soil layers (A1 and A2 layers) had higher pH at the INSIDE and DRIPLINE regions of Dacrydium than at those of Lithocarpus. In contrast to the INSIDE and DRIPLINE regions, the effects of tree species on soil pH and the concentration of condensed tannins were not signicant at the OUTSIDE region. Organic C, total N, C-toN ratio, and total phenolics did not show a distinct spatial pattern depending on tree species or distance. As expected, soil layer (i.e., depth) was always strongly related to all soil physicochemical properties analyzed (Table 1). 3.2. The spatial pattern of the composition of the soil microbial community The primary difference in the soil microbial community between Dacrydium and Lithocarpus was in fungi-to-bacteria ratio (Table 1 and Fig. 1). The fungi-to-bacteria ratio was higher for Dacrydium than for Lithocarpus, and the difference was signicant at the INSIDE region (Table 1). In contrast to the effect of tree species on fungi-to-bacteria ratio, tree species and distance from a tree trunk did not have signicant inuences on the gram-positive bacteria to gram-negative bacterial ratio (Table 1). The overall composition of soil microbial community was analyzed with PCA, and only the rst two components are reported here. PC1 and PC2 explained 26.5% and 13.1% of total variation, respectively. The effects of tree species on PC1 was signicant only at the INSIDE region, whereas the effects of tree species on PC2 were signicant in the all three regions (Table 1). As expected, soil layer showed the strongest effects on soil microbial composition (Table 1), and each layer formed a distinct cluster on the PCA ordination (Fig. 2a). The dissimilarity of the composition of soil microbial communities between the two tree species decreased with increasing distance from a tree trunk (Fig. 2b). The mean

Fig. 1. Fungi-to-bacteria ratio of soil samples beneath each tree species. Bars indicate SEM. Layer abbreviations and symbols mean the same as in Materials and Methods. Fungi-to-bacteria ratios were calculated as 18:2u6,9/(sum of all bacterial lipid).

values of dissimilarity were greater in the O layer than in the A layer at the INSIDE and DRIPLINE regions, yet the differences were not statistically signicant (Fig. 2b). 3.3. The spatial pattern of soil enzyme activities The activities of b-D-glucosidase and phenol oxidase in INSIDE and DRIPLINE regions were signicantly different between Dacrydium and Lithocarpus (Table 1). The mean activities of

Table 1 Effects of tree, distance from tree trunk, and layers on soil physicochemical properties, lipid abundance, and enzyme activities. Distance Effects INSIDE Tree Layers 226**** 62.0**** 103**** 168**** 15.3*** 148**** 772**** 167**** 243**** 85.0**** 0.8 284**** 22.5**** 223**** 69.9**** 28.2**** 29.7**** Interaction 1.35 8.0* 4.0y 0.001 0.01 0.25 1.88 0.54 0.03 2.0 0.7 5.87* 4.05y 0.002 2.4 5.47* 6.19* DRIPLINE Tree 2.81 7.8* 2.3 0.37 0.61 0.19 0.06 4.90y 2.49 0.6 2.1 3.04 4.80y 1.36 8.5* 30.7** 0.93 Layers 152**** 52.1**** 68.1**** 188**** 14.4*** 119**** 415**** 80.5**** 215**** 127**** 1.6 264**** 26.0**** 234**** 36.5**** 1.07 31.2**** Interaction 0.80 1.0 1.1 0.02 0.03 0.00 0.03 0.08 1.43 3.4y 0.6 2.55 2.59 0.46 5.5* 10.1** 0.59 OUTSIDE Tree 0.002 1.1 1.50 0.20 0.00 0.26 0.29 0.19 0.29 0.2 0.1 3.00 7.24* 0.07 0.00 0.02 3.44 Layers 193**** 89.3**** 110**** 161**** 13.4*** 150**** 613**** 75.2**** 221**** 82.1**** 0.6 147**** 16.1*** 157**** 29.6**** 1.53 43.1**** Interaction 0.01 0.2 0.63 0.01 0.00 0.02 0.003 0.001 0.13 0.3 3.0y 1.87 2.72 0.00 0.00 0.03 0.59

Soil physicochemical properties Soil moisture 1.82 5.0y pH (H2O) pH (KCl) 3.3 Organic C 0.04 Total N 0.01 C/N ratio 0.25 Total phenolics 0.90 Condensed tannins 13.72** Soil microbial community Total lipid Fungi/bacteria ratio Gm/Gm- ratio PC1 (26.5%) PC2 (13.1%) Soil enzyme activity Acid phosphatase beD-glucosidase Phenol oxidase Peroxidase 3.74 6.0* 0.8 8.11* 11.82* 0.35 10.2* 12.7* 2.25

Values indicate F-value. Bold values indicate signicant effects at yP < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, respectively.

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a b

Fig. 2. The composition of soil microbial communities (a) and composition dissimilarity between Dacrydium and Lithocarpus (b). Dissimilarity is assessed by calculating the Euclidean distances between the values of PCs of Dacrydium and those of Lithocarpus for each sampling category. Different upper-case letters indicate the signicant difference in the mean values of dissimilarity between distance regions. Permutation test (999 permutations), P < 0.05.

b-D-glucosidase and phenol oxidase were generally lower in soil of

Dacrydium than in that of Lithocarpus (Table 2). The mean activity of b-D-glucosidase in soil beneath Lithocarpus was higher at the INSIDE region than at the OUTSIDE region, but this was not the case for Dacrydium. The activity of acid phosphatase was not signicantly different between Dacrydium and Lithocarpus, although we previously found that the acid phosphatase activity was signicantly higher beneath Dacrydium (Ushio et al., 2010). We did not nd signicant inuences of tree species on the activities of peroxidase at the any distance regions. 3.4. Overall relationships among soil enzyme activities, physicochemical properties and microbial community composition RDA revealed the relative contributions of soil physicochemical properties and soil microbial community (i.e., lipid principle components) to the soil enzyme activities (Fig. 3). Here, we report only the rst two components of four RDA axes. Axis1 and axis2
Table 2 Mean values of soil enzyme activities beneath each tree species. Soil layer Dacrydium gracilis (conifer) INSIDE Acid phosphatase (mmol g1 h1) O1 O2 A1 A2 O1 O2 A1 A2 O1 O2 A1 A2 O1 O2 A1 A2 23.9 (3.1) 16.2 (0.9) 3.4 (0.2) 2.0 (0.4) 0.369 (0.061) 0.155 (0.030) N.D. N.D. 1.170 0.822 0.580 0.213 0.799 1.612 3.083 2.137 (0.440) (0.120) (0.182) (0.094) (0.518) (1.044) (0.449) (0.483) DRIPLINE 22.6 (1.6) 16.1 (1.8) 4.2 (1.1) 1.6 (0.2) 0.229 (0.072) N.D. 0.008 (0.009) N.D. 0.082 0.446 0.452 0.394 0.270 1.070 3.000 2.507 (0.150) (0.061) (0.063) (0.088) (0.161) (0.554) (0.669) (0.793)

explained 14.7% and 8.1% of total variation in O layers, respectively. In A layers, axis1 and axis2 explained 23.9% and 12.8% of total variation, respectively. PC2, pH (H2O), condensed tannins and soil moisture had signicant inuences on soil enzyme activities in O layers (Fig. 3a). Microbial community composition (i.e., PC2), as well as soil physicochemical properties, signicantly explained the enzyme activities in O layers. Organic C concentration and pH (H2O) had signicant inuences on soil enzyme activities in A layers (Fig. 3b). In both soil layers, pH (H2O) had signicant inuences on soil enzyme activities. 4. Discussion 4.1. The spatial pattern of soil physicochemical and microbial properties associated with plant canopies We found a distinct spatial pattern of soil physicochemical properties, microbial composition, and enzyme activities associated with plant canopies in the tropical montane forest

Lithocarpus clementianus (broadleaf) OUTSIDE 22.6 (2.3) 18.8 (2.5) 3.5 (0.5) 1.9 (0.2) 0.392 (0.072) 0.139 (0.142) N.D. N.D. 0.621 1.716 0.447 0.291 (0.306) (1.507) (0.217) (0.103) INSIDE 24.1 (3.3) 14.2 (1.2) 2.9 (0.6) 1.8 (0.4) 0.658 0.459 0.159 0.060 3.152 2.705 0.940 0.744 (0.160) (0.109) (0.070) (0.028) (0.920) (0.578) (0.227) (0.078) DRIPLINE 20.3 13.5 3.1 1.8 0.452 0.306 0.096 0.033 1.113 1.171 0.794 0.582 0.703 0.847 3.040 3.893 (2.3) (1.6) (0.5) (0.2) (0.129) (0.069) (0.022) (0.036) (0.105) (0.263) (0.161) (0.170) (0.451) (0.562) (0.582) (0.570) OUTSIDE 23.0 (3.5) 17.4 (2.9) 3.4 (0.1) 1.8 (0.1) 0.320 (0.119) 0.247 (0.102) 0.017 (0.015) N.D. 0.846 0.803 0.463 0.437 0.456 0.428 2.731 4.137 (0.466) (0.266) (0.107) (0.134) (0.122) (0.257) (0.598) (1.109)

b e De
glucosidase (mmol g1 h1) Phenol oxidase (mmol g1 h1) Peroxidase (mmol g1 h1)

N.D. 0.100 (0.166) 1.465 (0.528) 2.603 (1.039)

N.D. N.D. 2.182 (0.730) 3.186 (0.300)

Parentheses indicate SEM. n 4.

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Fig. 3. Redundancy analysis among soil enzyme activities, selected soil physicochemical properties, and lipid principle components in O layer (a), and A layer (b). The concentrations of organic C (%), total N (%), total phenolics and condensed tannins are abbreviated as C, N, TP, and CT, respectively. Signicant explanatory variables are indicated by asterisks. Permutation test (999 permutations). *P < 0.05, **P < 0.01.

(Tables 1 and 2 and Figs. 1 and 2). Tree species had signicant inuences on soil pH (H2O), the concentration of condensed tannins, fungi-to-bacteria ratio, PC1, PC2, b-D-glucosidase activity and phenol oxidase activity at the INSIDE region (Table 1). The concentration of condensed tannins was higher beneath the tree crown of Dacrydium than beneath that of Lithocarpus (Table S1). A greater concentration of foliar condensed tannins in Dacrydium than in the other species (Suzuki, S. unpublished data) as well as the abundance of Dacrydium litter underneath Dacrydium canopies can explain the greater concentration of condensed tannins in the soils. We suggest that the condensed tannins derived from litter inuence the composition of soil microbial communities, enzyme activities and subsequent soil nutrient availability because of the highly reactive chemical traits of condensed tannins with proteins (Scalbert, 1991; Httenschwiler and Vitousek, 2000; Kraus et al., 2003; Ushio et al., 2009). Plant roots may also affect soil microbial communities and enzyme activities through secreting labile as well as recalcitrant organic matter (e.g., Lejon et al., 2005; Von Arnold et al., 2005), yet our current data set do not allow us to assess the inuences of plant roots. However, our results suggest that above-ground litter exerts predominant inuences on soils in this forest because soil physical and biological properties correspond spatially with canopy crowns. The composition of soil microbial communities between Dacrydium and Lithocarpus was most dissimilar at the INSIDE region (Fig. 2b). As PC1 and PC2 explained ca. 40% of total variation in microbial composition, tree species, distance from a tree trunk, and soil layer mostly explained the variation of microbial composition. The primary difference in the soil microbial composition between Dacrydium and Lithocarpus was in the fungi-to-bacteria ratio (Table 1 and Fig. 1). The difference in the concentration of condensed tannins in soil might have partly contributed to the difference in the fungi-to-bacteria ratio, because fungi are generally more tolerant to toxicity of condensed tannins than bacteria (Scalbert, 1991), and because fungi can utilize those recalcitrant substrates as an energy source (Scalbert, 1991). 4.2. Possible contributors to the spatial pattern of soil enzyme activities Soil enzyme activities were also different between Dacrydium and Lithocarpus. Beneath Dacrydium tree crowns, the activities of

carbon-degrading enzyme were generally low, whereas those of phosphorus-degrading enzyme were slightly (but not signicantly) higher (Table 2). The RDA suggests that soil pH, moisture and condensed tannins signicantly explains the variation of enzyme activities in O layers (Fig. 3a), which is in accord with previous studies (Kraus et al., 2003; Sinsabaugh et al., 2008). Thus, the differences in soil pH and condensed tannins between Dacrydium and Lithocarpus can be one of the mechanisms that contribute to the differences in the soil enzyme activities between the two tree species. The RDA also showed that lipid principle components (i.e., indicators of microbial composition) could also explain some of the variations of the soil enzyme activities (Fig. 3). Not only soil physicochemical properties but also the differences in microbial composition between the tree species (Fig. 2) can partly contribute to the spatial pattern of soil enzyme activity. Since enzyme activity intrinsically drives degradation of organic matter in soil, the inuences of microbial composition on the enzyme activity can affect decomposition process (e.g., nitrogen mineralization rate and soil respiration rate). Indeed, recent studies reported that the difference in microbial composition resulted in the difference of soil respiration rate or nitrogen mineralization rate (Zogg et al., 1997; Balser and Firestone, 2005; Strickland et al., 2009). 4.3. Implications for plantesoil feedbacks in this tropical montane forest Spatial patchiness of soil physicochemical and microbial properties was formed underneath the crown of inuential tree species in the surface of the organic soil layer. This spatial pattern may occur in other forests (Saetre and Bth, 2000; Wardle et al., 2008) although further studies are needed to conrm this. Subsequent processes of the plantesoil feedback effects are likely to occur within the spatial range of soil properties associated with plant canopy. Previous studies indicated that the difference of soil physicochemical and microbial properties resulted in the differences of soil nutrient availability. For example, Fierer et al. (2001) indicated that the addition of condensed tannins decreased soil nitrogen availability for plants (i.e., net nitrogen mineralization rate). Moreover, Balser and Firestone (2005) showed that the composition of soil microbial community could be one of the determinants of net nitrogen

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M. Ushio et al. / Soil Biology & Biochemistry 42 (2010) 1588e1595 Binkley, D., 1995. The Inuence of Tree Species on Forest Soils: Processes and Patterns. Agronomy Society of New Zealand Special Publication, Canterbuty, New Zealand. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purication. Canadian Journal of Biochemistry and Physiology 37, 911e917. Crews, T.E., Kitayama, K., Fownes, J.H., Riley, R.H., Herbert, D.A., Mueller-Dombois, D., Vitousek, P.M., 1995. Changes in soil phosphorus fractions and ecosystem dynamics across a long chronosequence in Hawaii. Ecology 76, 1407e1424. Fierer, N., Schimel, J.P., Cates, R.G., Zou, J., 2001. Inuence of balsam poplar tannin fractions on carbon and nitrogen dynamics in Alaskan taiga oodplain soils. Soil Biology and Biochemistry 33, 1827e1839. Frelich, L.E., Calcote, R.R., Davis, M.B., Pastor, J., 1993. Patch formation and maintenance in an old-growth hemlock-hardwood forest. Ecology 74, 513e527. 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mineralization rate. Thus, the spatial pattern of soil properties associated with plant canopy (i.e., both physiochemical and microbial properties) can induce a distinct spatial pattern of soil nutrient availability in the forest. Indeed, net nitrogen mineralization rate is lower beneath Dacrydium than beneath Lithocarpus (Ushio et al. unpublished data). Because the soil of the tropical montane forest contains inherently low concentrations of available nitrogen and phosphorus (Kitayama et al., 2000, 2004; Hall et al., 2004), and because plant nutrition in the forest could depend on the process of mineralization of soil organic matter, the plant-inuenced physicochemical and microbial properties of surface soil can have signicant inuences on plant nutrition beneath the tree crown. 4.4. Conclusions In conclusion, we found that different tree species have distinct inuences on the soil physicochemical and microbial properties beneath the tree crown, and the inuences might be largely due to the differences of litter chemistry (i.e., the concentration of condensed tannins). Therefore, the species-rich forest on the tropical mountain consists of spatially distinct units of soil properties associated with plant canopy, and the spatial pattern of soil properties can inuence ecosystem dynamics in the forest through effects on a plantesoil feedback. Acknowledgements We thank the two anonymous referees for valuable comments on the manuscript. We thank Dr. Rota Wagai for assisting with soil sampling and for fruitful discussions, Dr. Sizuo Suzuki for providing us with the data on leaf phenolics, Dr. Hiroko Kurokawa for providing us with the methods for phenolic quantication, and Dr. Shoko Sakai for valuable comments on the manuscript. We thank staffs of the Sabah Parks for their support and permission throughout the course of our research. We also thank Dr. Harry Read and Mr. Kevin Budsberg and members of the Balser Lab at the University of WisconsineMadison for their support with lipid analysis. This research was supported by a grant-in-aid (MESSC 19380010) to K.K. and in part by Global COE program A06 to Kyoto University. M.U. is supported by a JSPS Research Fellowship for Young Scientists (21-1526). Appendix. Supplementary material Supplementary material associated with this paper can be found, in the online version, at doi:10.1016/j.soilbio.2010.05.035 References
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