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Applied Soil Ecology 47 (2011) 111–118

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Applied Soil Ecology

journa l homepage: www.e lsev ier .com/ locate /apsoi l

Soil bacterial communities in native and regenerated perhumid montane forests

Yu-Te Lina, Kamlesh Jangidb, William B. Whitmanb, David C. Colemanc, Chih-Yu Chiua,∗

a Biodiversity Research Center, Academia Sinica, Nankang, Taipei 11529, Taiwanb Department of Microbiology, University of Georgia, Athens, GA 30602-2605, USAc Odum School of Ecology, University of Georgia, Athens, GA 30602-2602, USA

a r t i c l e i n f o

Article history:Received 23 June 2010Received in revised form 9 November 2010Accepted 10 November 2010

Keywords:Bacterial communityForest managementForest soil16S rRNA genes

a b s t r a c t

Forest management often results in changes in soil properties and microbial communities. In the presentstudy, we characterized differences in soil bacterial communities caused by forest management practicesusing 16S ribosomal RNA (rRNA) gene clone libraries. The communities were from a disturbed Chamae-cyparis (DCP) forest subjected to harvesting of snags and downed logs, a secondary Chamaecyparis (SCP)plantation subjected to harvesting of old-growth trees, and a secondary cedar plantation (SCD). Theseforests were compared to a nearby native Chamaecyparis (NCP) forest in a perhumid montane ecosystem.At this locality, the elevation is from 1500 to 2100 m a.s.l., the mean annual precipitation >4000 mm, themean annual temperature about 12 ◦C, and the soil pH <4. The phyla Acidobacteria and Proteobacteriapredominated among the three disturbed forest soil communities. Several diversity indices and rarefac-tion curves revealed that the diversity of the SCD community was higher than that of the DCP soils. Thediversity of the SCP community was intermediate. The bacterial diversity of the NCP community waslower than communities in the three disturbed forest soils. Analysis of molecular variance revealed thatthe bacterial community in SCD soils significantly differed from those in the three Chamaecyparis forestsoils. Some of the abundant operational taxonomic units (OTUs) significantly differed among the fourforest soils. Compared to the three disturbed forest soil communities, the NCP community was domi-nated by Proteobacteria, which accounted for more than half of the community. These results suggestthat the disturbance of forest harvesting and tree species conversion influence the composition of bac-terial communities in natural and disturbed forests and increase the diversity of the disturbed forest soilcommunity.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Tree harvesting and planting result in forest soil disturbance.Such disturbances could alter several characteristics of soil quality,including soil organic matter content, and result in quantitativeand qualitative changes in soil carbon and nitrogen pools, aswell as soil biological properties (Chen et al., 2004; Burton et al.,2007; Chatterjee et al., 2008). The effects of such disturbance onsoil microbial community structure, diversity, and biomass havereceived some attention (Hannam et al., 2006; Curlevski et al.,2010). However, most of the investigations were conducted in tem-perate forest ecosystems; little information exists regarding theeffects of forest conversion and harvesting on microbial commu-nities in tropical and subtropical soils. Here, we investigated soilbacterial communities from forests subjected to harvesting andadjacent conifer plantations in a perhumid montane, subtropicalecosystem.

∗ Corresponding author. Tel.: +886 2 2787 1068.E-mail address: bochiu@sinica.edu.tw (C.-Y. Chiu).

We examined the diversity and composition of the indigenoussoil bacterial communities in disturbed forests located near theYuanyang Lake (YYL) forest ecosystem in Taiwan. YYL is located inthe northeastern region of Taiwan. The site is a subtropical moun-tainous cloud forest and experiences heavy cloud and frequent fog.This locality is temperate, very wet, and has high soil acidity. It is anuncommon ecosystem for a monsoonal part of Southeast Asia andconsists of forest dominated by Chamaecyparis tree species. Becauseof their valuable wood, these trees have long been a major har-vested timber species. Our previous study conducted in the nativereserve region indicated that the diversity of the soil bacterial com-munity was lower than that in other temperate and tropical forests.Proteobacteria, Acidobacteria and Actinobacteria predominate in thecommunity (Lin et al., 2010). However, the microbial communityin the disturbed forest soils adjacent to the YYL natural forest is stillunknown. Considering the disturbances inherent in establishingand harvesting plantations, we assumed that the composition anddiversity of the soil bacterial communities in these forests woulddiffer even though they are in the same climate and soil type.

From analysis of 16S ribosomal RNA (rRNA) clone libraries, weelucidated the effects of timbering on the soil bacterial commu-

0929-1393/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.apsoil.2010.11.008

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Table 1Soil chemical and physical properties of study sites.

Study site Elevation (m) pH Organic C(g kg−1)

Total N(g kg−1)

C:N

SCDa 1600 3.7 145 8.5 17.0SCPb 1600 3.6 475 18.9 25.1DCP 1500 3.7 432 22.7 19.0NCP 1950 3.5 483 22.5 21.5

a Data of SCD and NCP were from Imberger and Chiu (2002). Values of the NCPsite were the average of the data of summit and foot slope sites.

b Data of SCP and DCP were from Kang (2008).

nities among two disturbed Chamaecyparis forests and a coniferplantation. In the present study, we re-analyzed data from a pre-vious study (Lin et al., 2010), including sequences from foot slope(YYFS) and summit (YYSM) soils in natural Chamaecyparis forestsat YYL, in conjunction with new data. This study helps unravel theimpact of forest harvesting and conversion on the soil bacterialcommunities in this ecosystem.

2. Materials and methods

2.1. Study sites and soil sampling

This study was conducted in three disturbed forests locatednear a natural Chamaecyparis (NCP) forest dominated by Taiwanfalse cypress (Chamaecyparis formosensis) in the YYL ecosystem(24◦35′N, 121◦24′E). The elevation was 1500–1600 m a.s.l., themean annual precipitation >4000 mm, and the average annual tem-perature approximately 12 ◦C.

Two disturbed Chamaecyparis forests included a secondaryChamaecyparis (SCP) forest subjected to harvesting of old-growthtrees about 50 years ago and a disturbed, old growth Chamaecy-paris (DCP) forest, which had undergone harvesting of dead andfallen trees about 15 years ago. Timber of Chamaecyparis is rich withessence oil, which enables the wood to resist decay for decadeseven after the trees have fallen by occasional strong typhoons.Large numbers of fallen trees remained intact on the floor of thenative Chamaecyparis forest. Collecting fallen trees is consideredan alternative way to harvest these valuable trees and minimizedisturbance to the forest.

The third forest was replanted about 30 years ago with a purestand of Japanese cedar (Cryptomeria japonica) (SCD). Consideringthe impact of human activities in these forests, SCD was subjectedto the heaviest disturbance, then the SCP and DCP forests.

The soils in the three sampling sites were Inceptisol (Drys-trochrept) (Imberger and Chiu, 2002). The soils were stronglyacidic, with pH values of the surface soils ranging from 3.6 to 3.7.Other characteristics of soil sites are in Table 1.

Soil samples were collected in SCD, SCP and DCP forests. Foreach of the three sites, four replicates were collected from four50 m × 50 m plots. Soil samples 8 cm in diameter and 10 cm deepwere collected with use of a soil auger. Four subsamples collected ineach plot were combined. Visible detrital material, such as roots andlitter, were manually removed before material was passed througha 2-mm sieve. Soils were then stored at −20 ◦C for a few days beforeDNA extraction.

2.2. 16S rRNA gene clone library construction

The construction of 16S rRNA clone libraries was as describedpreviously (Lin et al., 2010). In brief, soil community DNA wasextracted by use of the PowerSoilTM DNA Isolation kit (MoBioIndustries) in accordance with the manufacturer’s instructions. Thebacterial 16S rRNA genes were amplified by PCR with the primerset 27F and 1492R (Lane, 1991). The PCR products were cloned by

use of the TOPO TA cloning kit (Invitrogen) and the pCR2.1 vec-tor. White colonies on selective Luria-Bertani (LB) agar plates wereplaced into 96-well blocks containing 1 ml LB broth plus kanamycin(50 �g ml−1) and grown overnight. Sterile glycerol was added to afinal concentration of 10%, and an aliquot of this was transferredto a 96-well sequencing block. Both the sequencing and originalculture blocks were stored at −80 ◦C.

2.3. DNA sequencing and taxonomic assessment

Bacterial clones were partially sequenced with the primer 27F.Sequence analysis involved use of an ABI PRISM Big Dye Termi-nator cycle sequencing ready reaction kit (Applied Biosystems)and an ABI 3730 Genetic Analyzer (Applied Biosystems) follow-ing the manufacturer’s instructions. Sequences were analyzedby use of the Mallard (Ashelford et al., 2006) and Pintail pro-grams (Ashelford et al., 2005) to test for chimeras. The sequenceswere taxonomically identified by use of the naive Bayesian rRNAclassifier (Wang et al., 2007) in the Ribosomal Database Project(http://rdp.cme.msu.edu/index.jsp).

Sequences were named as follows. For instance, the sequenceSCD101 comprised a six-character code that signified the sourceof forest soils (SCD, SCP or DCP), the sample replicate in the firstnumber (1 in this case), and a two-digit unique indicator of thesequence (01 in this case).

2.4. Phylogenetic dendrogram construction

The obtained clone sequences were submitted to the GenBankdatabase under the accession numbers HQ264404–HQ265229. TheNCP bacterial community was described previously (Lin et al., 2010)and was included for comparison purposes. The sequences werescreened against those in the NCBI GenBank database by use ofthe BLAST program. Phylogenetic relationships were analyzed asdescribed (Lin et al., 2010). Phylogenetic trees were constructed bythe neighbor-joining method (Saitou and Nei, 1987) with PHYLIPv3.6 (Felsenstein, 2005). Bootstrap values (Felsenstein, 1985) werecreated with 100 replicates.

2.5. Diversity estimates and library comparison

Distance matrices calculated by use of DNADIST in PHYLIPwere used as the input file for the program DOTUR (Schlossand Handelsman, 2005). Rarefaction analyses, richness, evenness,Shannon diversity index (H) and Chao1 estimates were calcu-lated for operational taxonomic units (OTUs) with an evolutionarydistance (D) of 0.03 (or about 97% 16S rRNA gene sequence similar-ity). To analyze the distribution of abundant taxa within libraries,groups were constructed by use of DOTUR at a distance of ≤0.03.These groups were then analyzed by the Fisher exact test (Agresti,1992). UniFrac (Lozupone et al., 2006) analysis was used to com-pare the clone libraries based on the phylogenetic information. TheUniFrac Significance test option with 100 permutations was usedto test the significant difference of each pair of samples. The Jack-knife Environment Clusters were used with the weighted algorithm(which considers relative abundance of OTUs) and the normaliza-tion step.

A hierarchy of log-linear models was used to examine differ-ences in the taxonomic composition among rRNA gene libraries(Jangid et al., 2008). The models sought to explain phylotype occur-rence as a function of treatment and were run separately withuse of the GENMOD procedure in SAS (SAS Inst., Cary, NC). Theresponse variable (i.e., separate counts for each phylotype fromreplicate libraries) was assumed to approximate a Poisson distribu-tion. The null model assumed that the distribution of all phylotypesacross all experimental conditions was the same, subject to sta-

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Table 2Phylotypes of clones in 16S rRNA gene libraries.

Phylogenetic group Clone library (% of clones)a Total

SCD SCP DCP NCPb

Acidobacteria 47.5 35.8 36.9 19.1 33.9Actinobacteria 3.5a 8.4b 8.2b 15.5b 9.3Bacteroidetes 3.5 4.0 0.7 0.6 2.1Chloroflexi 1.2 4.7 4.4 0.0 2.5Firmicutes 2.7 0.7 0.7 0.3 1.0Gemmatimonadetes 2.3 0.7 0.3 0.3 0.9Nitrospira 0.4 0.0 0.0 0.0 0.1Planctomycetes 3.9 6.0 3.4 4.9 4.6Proteobacteria 29.0 31.4 36.2 55.0 38.8

�-Proteobacteria 17.4 14.6 18.8 11.2 15.3�-Proteobacteria 3.9a 4.7a 4.8a 35.9b 13.4�-Proteobacteria 3.9 6.6 6.5 7.3 6.1�-Proteobacteria 3.5ab 5.5a 6.1a 0.6b 3.8Unclassified Proteobacteria 0.4 0.0 0.0 0.0 0.1

Verrucomicrobia 0.4 2.2 2.4 1.5 1.6Unclassified bacteria 5.8 5.8 6.8 2.7 5.2Total clone numbers 259 274 293 329 1155

a Phylotypes with the same letter in each row indicates no significant differencein abundance according to the GENMOD procedure at p < 0.01.

b Data from Lin et al. (2010).

tistical variability. Model selection and calculation and correctionfor overdispersion were described previously (Jangid et al., 2008).Because the number of replicates for the NCP site was larger thanthat for the other sites, six versus four, the analysis was repeatedthree times with random selections of four of the six NCP replicates.All analyses yielded similar conclusions.

3. Results

3.1. Phylogenetic groups represented in the clone libraries

About 40–130 clones of 16S rRNA genes were sequenced fromeach replicate sample collected from the three disturbed forestsoils. Each site was represented by four replicate samples, and thesequences from replicates of each site were then combined for fur-ther analyses. We obtained 259 clones for the SCD site, 274 for SCPand 293 for DCP (Table 2). No chimeric sequences were detected,and all the resulting sequences were used for further analysis. Allclones were classified into 12 phylogenetic groups (Table 2). Inthree study sites, the most abundant groups were Acidobacteria andProteobacteria, comprising about 70% of all clones. Actinobacteriawas the third most abundant group. The remaining phyla presentwithin the libraries, such as Planctomycetes and Chloroflexi, all com-prised less than 5% of the clones. In addition, about 6% of clones fromthree disturbed forest soils were only distantly related to culturedbacteria and were designated as the unclassified bacterial group.These clones included a representative of the deeply branching butso far uncultured groups, Genera incertae sedis OP10. In the libraryfrom NCP soils, the most abundant groups were Proteobacteria, Aci-dobacteria and Actinobacteria, which accounted for about 90% oftotal clones (Table 2).

Despite the similarity of the relative abundance of clones affili-ated with various bacterial groups in all the soils, some phylotypesshowed differences detected by the hierarchy of log-linear mod-els. The abundance of �-Proteobacteria was significantly higher inthe NCP than other soils (Table 2). In contrast, the abundance ofthe unclassified group was significantly lower in the NCP than inother soils. The abundance of �-Proteobacteria was also significantlylower in the NCP than SCP and DCP soils. Although the abundance ofthis phylotype in SCD soils was similar to that in SCP and DCP soils,it was not significantly different from that in NCP soils at p = 0.01.Similarly, the abundance of Actinobacteria was significantly lowerin SCD than NCP soils. Although the abundance of Actinobacteria

in SCP and DCP soils was similar to that in SCD soils, it was notsignificantly different from that in NCP soils at p = 0.01. The meanabundance of Acidobacteria and �-Proteobacteria varied two-foldwithin the soils, but these differences were not significantly dif-ferent because of the high variation among the replicate samples(Table 2). Thus, despite the similar abundance of phylotypes in allsoils, most differences were observed between the native NCP andthe most heavily disturbed soil.

In the Acidobacteria, clones were further classified into subdivi-sions 1, 2, 3, 4, 5, 6, 13 and 14 (Fig. 1) according to previous studies(Hugenholtz et al., 1998; Barns et al., 2007). Most of the sequences(82%) were in subdivisions 1 and 2. Within the �-Proteobacteria,an abundant OTU with 22 sequences was related to Bradyrhizo-bium spp. (Fig. 2). Other bacterial taxa closely related to the soilclones included Acetobacteraceae, Caulobacteraceae and Rhodospir-illales spp. (Fig. 2). About 10% of clones were affiliated with anunclassified �-proteobacterial group.

3.2. Diversity of soil bacterial communities

For calculation of diversity indices, OTUs were formed atD ≤ 0.03 (about 97% sequence similarity). From the richness andChao1 estimator, the diversity of the bacterial community washigher in SCD soils than in SCP and DCP soils (Table 3). This con-clusion was supported by rarefaction curve analysis. The failure ofthe rarefaction curves to plateau indicated that these communi-ties were incompletely sampled (Fig. 3). Nevertheless, the slopesof curves for SCP and DCP soil communities were less steep thanthat for SCD soils, which suggests that the diversity was higher inSCD soils (Fig. 3). The curves also revealed that the community wasmore diverse in SCP than DCP soils, which is similar to the rich-ness and Chao1 estimator results. To gain further insight into therelative diversity of these communities, the Chao1 estimator wascalculated at different sample sizes (Kemp and Aller, 2004). Becausethe estimators approached a stable plateau with increasing samplesize (data not shown), they appeared to be reliable indicators ofdiversity. Analysis of the sizes of abundant OTUs provided a ratio-nale for the high diversity of the SCD soil community. In the SCPand DCP soils, 28% and 19% of the clones, respectively, were insingle-member OTUs or singletons. For the SCD soils, 38% of theclones were in singletons. In SCP and DCP soils, 22% and 25% of theclones were members of abundant OTUs (see below). In SCD soils,the abundant OTUs accounted for 20% (Table 4). In addition, fromthe richness, Chao1 estimator and rarefaction curve analysis, thethree disturbed forest soil communities were all more diverse thanthe NCP community (Table 3; Fig. 3).

3.3. Abundant OTUs in soil bacterial communities

Analyses of composition differences of communities alsorevealed the distinctness of the SCD and Chamaecyparis forest soils.UniFrac Significance analyses were used to compare the bacterialcommunities on the basis of the phylogenetic information. Thep-values revealed that the SCD community was significantly dif-ferent from the Chamaecyparis forest soil communities (Table 5).After removing the most abundant �-proteobacterial OTUs, the foursoil communities were not significantly different (data not shown).Thus, the differences among the ocommunities might result largelyfrom the abundance of �-Proteobacteria. In the cluster analyses ofthe clone libraries, the SCD community was well separated fromthe cluster formed by the SCP, DCP and NCP communities (Fig. 4).

Some of the differences in composition were identified by exam-ination of the abundant OTUs or those with sizes larger than 10,which represented about 31% of all clones (Table 4). Because repre-sentatives of each OTU were obtained from independent replicatesin multiple sampling locations and the representation was similar

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Fig. 1. Phylogeny of representative Acidobacteria clones for each OTU. OTUs were formed at evolutionary distances ≤0.03. Clones derived in this study are given in boldface.Subdivisions are indicated by brackets. The number of sequences for each OTU is in parentheses following the clone name and GenBank accession number. The names of thesequences comprise a six- to seven-character code that signifies the source from three disturbed forests (two Chamaecyparis forests, SCP and DCP, and one cedar plantation,SCD), and other numbers as described in the Methods. Numbers at nodes denote bootstrap confidence values obtained with 100 resamplings; values ≤70 are not shown. Thescale bar indicates 0.1 substitutions per nucleotide position.

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Fig. 2. Phylogeny of representative �-Proteobacteria clones. Notation is as described for Fig. 1.

in the different sample replicates (data not shown), their abun-dance was not due to PCR or cloning artifacts or to a single unusualsample. The most abundant OTU was from the Proteobacteria andrelated to the genus Burkholderia. It was mainly present in the

NCP community. The distribution of the abundant acidobacterialOTUs, such as DCP110, SCP119 and SCD164, was significantly dif-ferent among the four soil communities by the Fisher exact test(Table 4). As compared with disturbed forest soil communities, in

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Table 3Diversity indices for the soil bacterial communities as represented in the 16S rRNAgene librariesa.

Index SCD SCP DCP NCPb

Sc 148 147 125 95Nd 259 274 293 329Evennesse 0.98 0.98 0.98 0.97Richnessf 0.38 0.28 0.18 0.14Shannong 0.98 0.98 0.98 0.97Chao 1 338 (257, 480)h 215 (185, 268) 166 (146, 205) 165 (128, 244)

a Calculations were based on OTUs formed at an evolutionary distance of <0.03(or about 97% similarity).

b Values were calculated from Lin et al. (2010).c S defined as the number of OTUs observed.d N defined as the number of sequences.e Evenness defined as H/log S. The observed/maximum possible value is reported.f Richness = (number of singleton OTUs − 1)/log N. The maximum value is

(N − 1)/log N. The observed/maximum possible value is reported.g Shannon diversity index (H). The observed/maximum possible value is reported.h Confidence intervals (95%) for the Chao1 estimator are shown in parenthesis.

0

60

120

180

350280210140700

Number of Sequences Sampled

Num

ber

of O

TU

s O

bser

ved

Fig. 3. Rarefaction curve analysis for secondary cedar plantation (�), secondaryChamaecyparis (�), disturbed Chamaecyparis (�), and native Chamaecyparis (©) for-est soil libraries. OTUs were defined as sequences sharing 97% nucleotide sequencesimilarity. Values of NCP were calculated from data in Lin et al. (2010).

Table 4Distribution of the most abundant OTUs in 16S rRNA gene clone libraries.a

Clone nameb Taxonomic affiliation Clone library Nc

SCD SCP DCP NCPd

YYSM113* Burkholderia (AJ292637) 3b 4b 0b 90a 97DCP110* Acidobacteria (FJ466150) 5c 4c 16a 0b 25SCD160 Acidobacterium (FJ625367) 10 3 5 4 22SCP431 Bradyrhizobium (FJ418920) 3 7 7 5 22SCD306 Acidobacteria (FJ624909) 7 7 2 5 21SCD136* Rhodoplanes (FJ475449) 5ab 1b 5ab 8a 19SCP135 Acidobacteria (EU680446) 5 6 6 1 18SCP117 Acidobacterium (FJ466282) 2 6 5 5 18YYSM560* Burkholderia (GU472987) 0b 0b 0b 16a 16SCP243 Steroidobacter (AY963328) 1 6 5 3 15SCP275 Acidobacteria (EU680344) 1 5 4 2 12SCD164* Acidobacteria (EU445202) 5a 2ab 5a 0b 12DCP138 Actinomadura (EF220691) 0 2 5 5 12DCP340* Mycobacterium (EU138967) 0b 1b 2ab 9a 12SCD253* Acidobacterium (FJ625317) 2ab 0b 0b 9a 11SCP127* Caulobacter (FJ936950) 0b 2ab 0b 9a 11DCP118 Actinomadura (FJ669088) 0 1 4 5 10SCD368* Acidobacteria (FJ466178) 4a 3ab 3ab 0b 10

a OTUs formed at an evolutionary distance ≤0.03 (or about >97% similarity). Datawith the same letter in each row indicates no significant difference according toLSD-test at p < 0.05.

b Representative clone for each OTU. OTUs with nonrandom distribution aremarked by an asterisk.

c Total number of clones in an OTU.d Data were calculated from Lin et al. (2010).

Table 5Statistical significance (p-values) of differences among the forest soil bacterialcommunities.

SCD SCP DCP NCPa

SCD – <0.001b <0.001 <0.001SCP – 0.43 0.21DCP – 0.04NCP –

a Values were calculated from Lin et al. (2010).b p-Values of UniFrac significance test were based on 100 permutations. Bold

values indicate significant difference at p < 0.05.

NCP soils, some abundant OTUs, such as �-, �-proteobacterial andactinobacterial-affiliated OTUs, were absent or in low proportionsin the three disturbed forest soils (Table 4).

4. Discussion

Our results reveal that the bacterial diversity varied with dif-ferent degrees of disturbance in three forest ecosystems in theYYL region in Taiwan. The community in SCD soils with the heav-iest disturbance was the most diverse, then those in SCP andDCP soils. Moreover, the community composition of disturbed for-est soils differed greatly from that in natural forest soils in thisregion. Proteobacteria accounted for more than half of the totalclones, and �-Proteobacteria was the most dominant group in NCPsoils; in contrast, �-Proteobacteria was the most abundant groupin the three disturbed forest soils. Differences in soil bacterialcommunities have been detected when comparing undisturbedforests and pastures established from cleared forest land (Nüssleinand Tiedje, 1999). Proportional differences in �-Proteobacterial-affiliated clones were also found between forests with whole-treeharvesting without soil compaction and those with harvesting pluscomplete surface organic matter removal with heavy soil com-paction (Axelrood et al., 2002). The harvesting and replantationin these three disturbed forests caused the loss of shading over-story, and the exposure to sunlight might lead to soil temperaturefluctuation and water loss. Increased availability in NH4

+ has beenreported for clear-cut treatments (Smith et al., 2008). Differencesin nitrogen input could affect organic matter decomposition andthus result in microbial community and/or decomposing-enzymeshifts (Janssens et al., 2010). Soil C-to-N ratio and the response oftrees to this ratio all influence soil microbial community composi-tion (Högberg et al., 2007). Forest management could also influenceseveral environmental factors simultaneously. Thus, discriminat-ing the effects of an individual factor on the community is difficult.

In this study, the soil bacterial communities in the three dis-turbed forests were all more diverse than that in natural forest soils.The disturbed forests experienced more management than the NCPforest, which is a natural reserve with limited anthropogenic dis-turbance. Previous studies conducted in Georgia, USA, also showedthat bacterial communities in frequently tilled cultural soils were

0.1

SCD

NCP

DCP

SCP

100

100

Fig. 4. A dendrogram from UniFrac Jackknife Environment Clusters analysis of 16SrRNA gene clone libraries. Analysis involved weighted data. Numbers at nodes indi-cate the frequency with which nodes were supported by jackknife analysis.

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more diverse than in less disturbed forest soils (Jangid et al., 2008;Upchurch et al., 2008). The higher diversity in these more disturbedsoil bacterial communities also supported this conclusion. Besidesthe effect on diversity, the disturbance of tree conversion (Lauberet al., 2008; Curlevski et al., 2010) in SCD soils could have influencedthe soil bacterial community.

Molecular surveys have found Acidobacteria in a wide varietyof environments (Barns et al., 1999; Zimmermann et al., 2005;Janssen, 2006). In this study, Acidobacteria was also the dominantphylum in the community. However, these clones are not closelyrelated to known species of this phylum, Acidobacterium capsu-latum (Kishimoto et al., 1991), Geothrix fermentans (Coates et al.,1999) and Holophaga foetida (Liesack et al., 1994). Hence, predict-ing function from their properties is not possible. The Acidobacteriamight also be metabolically active as well as numerically dominantin soils (Lee et al., 2008). The functional diversity of Acidobacteriais important to examine further to elucidate their ecological roles.In addition, Acidobacteria are considered oligotrophs, so they canmaintain viability in nutrient-limited environments (Fierer et al.,2007). Hence, the abundance of Acidobacteria might indicate thelimited availability of nutrients or oligotrophic nature of the threedisturbed forest soils. By contrast, more than half of the clones wereProteobacteria in the NCP soil community. The �-proteobacterialclones were the most abundant group in the Proteobacteria. Incontrast to oligotrophic Acidobacteria, the �-Proteobacteria arecopiotrophic. Their relative abundance was highest in soils withhigh C availability (Fierer et al., 2007). Axelrood et al. (2002) showedthat Acidobacteria are generally less abundant and �-Proteobacteriamore abundant in soils with high concentrations of organic carbon.In NCP, the relative richness in soil organic carbon may create amore favorable habitat for the growth of �-Proteobacteria.

Among the three disturbed forest soil communities, �-Proteobacteria was the most abundant group of the Proteobacteria.One abundant �-proteobacterial OTU, represented by cloneSCP431, which was equally distributed throughout the study sites,is related to Bradyrhizobium spp. (Table 4). It was highly related tothe OTU NT21 H01, which was observed in agricultural and forestsoils in Georgia (Upchurch et al., 2008). The genus Bradyrhizobiumincludes slow-growing bacteria capable of nitrogen fixation andnodule formation on leguminous plants (Jordan, 1982). SymbioticBradyrhizobium strains have been isolated from nodules of highlydivergent legume tribes, including herbaceous and woody speciesof tropical and temperate origin (Menna et al., 2009). The physiolog-ical properties of the genus Bradyrhizobium indicate the potentialroles of this abundant OTU for symbiotic nitrogen fixation in naturaland disturbed forest ecosystems.

Because of the limited size of our clone libraries, only a por-tion of the soil diversity was sampled. For instance, more extensivesequencing efforts with pyrosequencing methodologies typicallyidentify thousands of OTUs in soil (Roesch et al., 2007; Acosta-Martínez et al., 2008). Nevertheless, the approach we describehas been widely used to study the compositional and structuraldifferences between soil microbial communities. The strategy ofreplicates for each treatment, community DNA extraction, PCRamplification, clone library construction and sequencing has beenused to successfully examine the effects of forest management inother soil microbial communities (He et al., 2006). However, moreextensive sampling of the soil microbial community, such as bypyrosequencing or other next-generation sequencing methodolo-gies, will be necessary to fully elucidate the changes in the bacterialcommunities in these soils.

5. Conclusions

Forest management practices, including harvesting and treeconversion, clearly influence the soil microbial community compo-

sition in natural and disturbed forests. The higher the disturbanceof forest management, the higher the diversity of the soil micro-bial community. The highly diverse and different composition ofcommunities in disturbed forests indicates that the bacterial com-munities are not fully restored even after 50 years of management.These findings significantly improve on our understanding of vari-ation in soil bacterial community in disturbed forests. Furtherinvestigation of the functional diversity of soil bacterial communi-ties in disturbed forest ecosystems is needed to address the impactof forest managements on ecosystem function.

Acknowledgement

This work was supported by the Taiwan National Science Coun-cil, Taiwan (NSC 97-2621-B-001-001-MY3).

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