Bacterial Community Composition in Brazilian Anthrosols and Adjacent Soils Characterized Using Culturing and Molecular Identification

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1 Micro Ecol (2009) 58:23 35 DOI 10.7/s y SOIL MICROBIOLOGY Bcteril Community Composition in Brzilin Anthrosols nd Adjcent Soils Chrcterized Using Culturing nd Moleculr Identifiction B. O Neill & J. Grossmn & M. T. Tsi & J. E. Gomes & J. Lehmnn & J. Peterson & E. Neves & J. E. Thies Received: 23 July 2008 /Accepted: 18 Mrch 2009 /Pulished online: 21 April 2009 # Springer Science + Business Medi, LLC 2009 Astrct Microil community composition ws exmined in two soil types, Anthrosols nd djcent soils, smpled from three loctions in the Brzilin Amzon. The Anthrosols, lso known s Amzonin drk erths, re highly fertile soils tht re legcy of pre-columin settlement. Both Anthrosols nd djcent soils re derived from the sme prent mteril nd suject to the sme environmentl conditions, including rinfll nd temperture; however, the Anthrosols contin high levels of chrcol-like lck cron from which they derive their drk color. The Anthrosols typiclly hve higher ction The uthor J. Peterson is lredy decesed. B. O Neill : J. Grossmn : J. Lehmnn : J. E. Thies Deprtment of Crop nd Soil Sciences, Cornell University, 235 Emerson Hll, Ithc, NY 14853, USA M. T. Tsi : J. E. Gomes Centro de Energi Nucler n Agricultur, Cix Postl 96, Av. Centenário no. 303, CEP: Pircic, São Polo, Brzil J. Peterson University of Vermont, Burlington, VT 05405, USA E. Neves Museo de Arqueologi e Etnologi, Universidde de São Pulo, São Pulo, SP , Brzil J. E. Thies (*) Deprtment of Crop nd Soil Sciences, Cornell University, 722 Brdfield Hll, Ithc, NY 14853, USA e-mil: jet25@cornell.edu Present Address: J. Grossmn Deprtment of Soil Science, North Crolin Stte University, PO Box 7619, Rleigh, NC , USA exchnge cpcity, higher ph, nd higher phosphorus nd clcium contents. We used culture medi prepred from soil extrcts to isolte cteri unique to the two soil types nd then sequenced their 16S rrna genes to determine their phylogenetic plcement. Higher numers of culturle cteri, y over two orders of mgnitude t the deepest smpling depths, were counted in the Anthrosols. Sequences of cteri isolted on soil extrct medi yielded five possile new cteril fmilies. Also, higher numer of fmilies in the cteri were represented y isoltes from the deeper soil depths in the Anthrosols. Higher cteril popultions nd greter diversity of isoltes were found in ll of the Anthrosols, to depth of up to 1 m, compred to djcent soils locted within m of their ssocited Anthrosols. Compred to stndrd culture medi, soil extrct medi reveled diverse soil microil popultions dpted to the unique iochemistry nd physiologicl ecology of these Anthrosols. Introduction Amzonin drk erths (ADE) contin orgnic mendments, such s chrcol, tht were incorported into them in pre- Columin times. This hs resulted in soils prized for their sustined fertility. The existence of these Anthrosols in close proximity to djcent, nutrient-poor soils formed from the sme prent mterils provided n opportunity to study vriles, other thn their inherent minerlogy, tht influence their cteril diversity. Cloning nd sequencing hve reveled distinctly different cteril communities in the Anthrosols compred to djcent soils [22], ut the fctors controlling these differences nd the unique roles tht microil communities my hve in ADE re not well-understood [41]. The high levels of lck cron (BC) present in ADE hve existed for severl hundred to thousnds of yers since

2 24 B. O Neill et l. their incorportion, in spite of intense wethering nd conditions highly fvorle for microil decomposition [20]. Soil temperture, moisture, texture, cron (C) content, nutrient vilility, nd ph, long with lnd use history, plnt species, nd sesonlity re mong the fctors tht hve een shown to ffect soil microil community composition [6, 7, 17, 49]. ADE hve higher concentrtions of key nutrients, such s phosphorus (P), clcium (C), nd soil orgnic C, ut lower respirtion rtes (cron dioxide evolution) compred to djcent soils (Ling, Ph.D. thesis). Understnding the interction of diverse microil popultions with the mny dynmic iogeochemicl fctors in soil remins gret chllenge [18, 21, 45, 49] nd is centrl to modeling rtes of nutrient turnover in soil ecosystems [2]. A key question for nutrient cycling in ADE is whether the pool of BC itself is cycling t gretly reduced rte or if the presence of BC hs ltered nutrient nd C cycling throughout the soil ecosystem. Furthermore, while soil horizon depth dicttes the volume in which key microil processes predominte [3], especilly in tropicl soils, the incresed fertility t depth in the ADE my gretly expnd the extent of microil ctivity relevnt to modeling nutrient dynmics. To compre soil microil communities in Anthrosols nd djcent soils, we used the differences in soil chemicl composition s the selective gent to isolte cteri. Bcteril culturing techniques cn e tedious nd revel only smll proportion of extnt orgnisms in soil, ut they re n excellent method for exmining the physiologicl nd metolic diversity of cteri potentilly ctive in soil ecosystems [23]. Comined with powerful moleculr techniques, culturing methods work synergisticlly to cpture nd identify distinct proportion of the microil diversity in soil nd more completely descrie its community composition [24, 45, 48]. In this study, we exmined cteril popultion size nd composition in pired tropicl soil types: the Anthrosols creted during pre-columin settlement nd djcent unmodified soils from three loctions in the Brzilin Amzon. Both soil types t ech loction were derived from the sme prent mteril nd were suject to the sme climte, ut differed y the presence/sence of BC nd the ssocited high P nd C contents in the Anthrosols. We cultured nd isolted cteri on miniml medium nd on soil extrct medi prepred from ech soil smple nd used colony polymerse chin rection (PCR) [34] to mplify 16S rrna genes from ech isolte [42]. Using restriction frgment length polymorphisms (RFLP) nd direct sequencing of 16S rrna, we identified nd compred the dominnt, culturle cteri residing in ech soil type t ech site t multiple soil depths. We hypothesized tht the Anthrosols would contin higher numers of culturle cteri in multiple soil horizons nd greter diversity of cteri nd tht the culturle cteril communities would e more similr mong the Anthrosols thn etween the Anthrosols nd the djcent soils smpled from the sme site. In comprison with djcent soils, etter understnding of the size nd diversity of the cteril popultion in ADE should inform future inquiry into the physiologicl ecology of BC-rich soils. Mterils nd Methods Site Descriptions nd Soil Smpling Soils were collected from three sites within the Amzon Bsin ech locted within 50 km of the city of Mnus, Brzil (3 08 S, W, 4 50 m ove se level). The nturl vegettion is tropicl lowlnd rin forest with men nnul rinfll from 2,000 to 2,400 mm [37]. The smpled soils were Anthrosols [29] nd their djcent, nturlly occurring, nutrient-poor soils, which re derived from tertiry sediments, primrily lterite. Three sites were smpled in this study: Hthr (Ht), Lgo Grnde (LG), nd Açutu (Acu). Horizon clssifiction in the Anthrosols ws determined y fctors such s density of pottery sherds, indictive of humn settlement. Smpled Anthrosols nd djcent soils were m prt t ech site nd were formed from the sme prent mteril. Anthrosols were smpled from existing rcheologicl excvtions y tking multiple uger cores within ech soil horizon nd compositing the cores. The unexcvted djcent soils t ech site were smpled y use of soil uger nd seprted y soil horizons. Multiple cores from ech horizon were mixed together. Approximtely 1.0 kg totl smple weight ws tken from ech horizon. Smples were stored in seled plstic gs t room temperture for 1 week nd then t 4 C for 12 weeks prior to culturing. Soil chrcteristics of pired A horizons t ech loction re presented in Tle 1. Medi Preprtion nd Culturing Soil extrcts were prepred from ech soil type, depth, nd site used in the culturing experiments. These extrcts were used to prepre gr-sed medi ccording to Burlge et l. [8]. To one prt of ech soil smple, two prts of douledistilled wter were dded (w/v) in milk dilution ottles. Soil slurries were sterilized for 1 h nd then llowed to settle nd cool to room temperture. Once cooled, the liquid soil extrcts were poured off nd frozen t 20 C. The soil extrcts were then thwed nd vcuum-filtered through 25-μm Whtmn no. 41 filter pper. For ech liter of soil extrct gr (SEA), 50 ml of soil extrct were dded to 950 ml distilled wter. Bcto Agr (1.8%, BD Difco, Frnklin Lkes, NJ, USA) ws dded nd the medium ws sterilized t 121 C for 20 min. Liquid soil extrct medi were prepred in the sme mnner nd used to nlyze

3 Microil Communities in Brzilin Anthrosols nd Adjcent Soils 25 Tle 1 Chemicl nd physicl chrcteristics of pired A horizons for Anthrosols nd djcent soils Grphitic BC (%) C/N CEC (mmol Chrge kg 1 ) C (mg g 1 ) P (mg g 1 ) N (mg g 1 ) Orgnic C (mg g 1 ) Cly (%) Silt (%) Snd (%) ph (1:2.5 H 2 O) Site Type Lnd feture Age (yers) 600 1, Ht Anth Under psture dense pot shrds , ,000 2, Adj Under erly secondry forest, deep ornge LG Anth Under secondry forest few pot shrds Adj Under secondry forest few concretions Acu Anth Ner g. field drk w/dense pot shrds Adj Under secondry forest yellow ltosol-like Arevited from Ling et l. [26] Totl N, P, nd C Percent grphitic BC out of totl orgnic C, determined y therml oxidtion mmonium N (NH 4 + N) nd nitrte (NO 3 N) contents on continuous flow utonlyzer (SEAL Anlyticl, Mequon, WI, USA). Elementl compositions of the soil extrct medi were determined y use of inductively coupled plsm spectroscopy (ICP, Spectro Ciros CCD, Kleve, Germny) t the Cornell Nutrient Anlysis Lortory in Ithc, NY, USA. The ph of the extrcts ws mesured for ll soil smples t the diluted concentrtions used for culturing. Solid R2A ws prepred using BD Difco (Frnklin Lkes, NJ, USA) dehydrted R2A medium (product no ). A modified liquid medium (R2L) ws prepred ccording to Fries et l. [19]. Sterile, molten R2A nd SEA were poured into gridded, 150-mm dimeter Petri pltes. Aliquots of 10 ml of R2L nd SEL medi (five replictes for ech of five dilutions per smple) were dded to 50 ml screw-top tues, cpped, nd utoclved for 15 min t 121 C. Soil inocul for culturing were prepred using 10 g soil, extrcted for 10 min t room temperture with gentle gittion in 90 ml sterile 0.1 M sodium phosphte uffer, ph 6.8. Five, tenfold, seril dilutions from ech soil smpled were prepred. Aliquots from ech dilution were used to inoculte: (1) Petri pltes contining SEA, (2) Petri pltes contining R2A, (3) five replicte tues of R2L, nd (4) five replicte tues of SEL for most prole numer (MPN) estimtions. Inoculted liquid nd solid medi were incuted in the drk t 30 C. For liquid cultures, growth (turidity) ws recorded iweekly for 120 dys, lthough no dditionl positive tues were oserved fter 42 dys. MPN estimtes were mde using the MPNES softwre [46]. Colonies formed on R2A nd SEA were counted dily over the first week nd iweekly therefter, with ny new colonies denoted y color-coding on the plte. During 4.5 months of incution (pproximtely 135 dys), pproximtely 20 colonies rising on SEA nd R2A were selected from pltes t multiple dilutions for ech smple (generlly three pltes out of five dilutions contined colonies t spcing suitle for single-colony isoltion). Colony selection ws sed on morphologicl differences nd the week in which the colony first ppered fter plting, with preference given to slowerforming colonies. Selected colonies were sucultured on the sme medium on which they were initilly cultured nd on the lternte medium (i.e., colonies rising on SEA were tested for growth on R2A nd those rising on R2A were tested for the ility to grow on SEA for ech soil extrct) nd growth noted fter incuting for up to 120 dys t 30 C in the drk. PCR-Restriction Frgment Length Polymorphism Anlysis Cells from purified colony isoltes (verified y microscopy) were used directly for DNA mplifiction y PCR using the forwrd primer 27f (5 -AGA GTT TGA TCC TGG CTC AG-3 ) nd reverse primer 1492r (5 -GGT TAC CTT GTT

4 26 B. O Neill et l. ACG ACT T-3 ) (Integrted DNA Technologies, Corlville, IA, USA), which trget the cteril 16S rrna genes [28]. Isolte DNA ws mplified using PTC therml cycler (MJ Reserch, Wlthm, MA, USA) nd the following progrm: 5 min t 94 C, followed y 35 cycles of 94 C for 45 s, 56 C for 45 s, nd 72 C for 1 min, nd finl extension step t 72 C for 10 min. Finl rection concentrtions were: 0.05 U µl 1 Tq polymerse (Promeg, Mdison, WI, USA), 1.0x PCR uffer supplied with the enzyme, 2.0 mm MgCl 2, 0.2 mm dntps, 0.1 µg µl 1 ovine serum lumin (BSA), oth primers t 0.1 µm, nd nuclese-free wter (Promeg). PCR products were verified y running them on 0.7% grose gels, stining the gel with SYBR Green I (Cmrex Biosciences, Est Rutherford, NJ, USA) nd visulizing the products using Fluor-S MultiImger (Bio-Rd, Hercules, CA, USA). Isolte mplicons ( 1,450 p) were then digested in 20 µl rection volumes using the restriction enzyme MspI (Promeg) t 10 U µl 1 with 1.0x uffer (supplied with the enzyme), 10 µg µl 1 BSA, nd nuclese-free wter. Restriction digests were crried out in PTC therml cycler (MJ Reserch) held t 37 C for 4.5 h with finl step t 70 C for 15 min to stop the rection s specified y the mnufcturer. Restriction frgments were seprted in 2% grose gels. Gels were stined with ethidium romide nd the RFLP ptterns visulized using the Fluor-S Multi- Imger (Bio-Rd). For cteril isolte RFLP fingerprints yielding the sme or similr nding ptterns using the MspI restriction enzyme, dditionl liquots of the 16S rdna mplicons were digested using the restriction enzymes RsI nd HhI (Promeg), ccording to the mnufcturer's protocols, nd the resulting RFLP fingerprints digitized s descried ove. Sequencing After riotype screening, 16S rdna mplicons of interest were clened prior to sequencing using ExoSAP-IT (USB, Clevelnd, OH, USA). Rections were crried out in PTC therml cycler (MJ Reserch) held t 37 C for 15 min followed y 80 C for 15 min to inctivte the rection. All sequencing rections were performed t the Cornell Biotechnology Resource Center in Ithc, NY, USA, using n Automted 3730X1 DNA Anlyzer (Applied Biosystems, Foster City, CA, USA). Sequences were deposited in the GenBnk nd ssigned the ccession numers EU EU Dt Anlysis A chimer check on the ligned sequences ws run using the Riosoml Dtse Progrm II, relese 8.1 [10]. Cluster nlysis of RFLP fingerprints ws crried out with the unweighted pir group method with rithmetic linkge method using nd presence/sence in the BioNumerics softwre (Applied Mths, Sint-Mrtens-Ltem, Belgium). Resulting inry dt were further nlyzed using JMP 5.1 (SAS Institute, Cry, NC, USA). Sequence lignment s well s phylogenetic nd moleculr evolutionry nlyses were conducted with MEGA version 3.1 [39], using the Jukes Cntor neighor-joining lgorithm. Distnce mtrices were constructed y the DNADIST progrm in PHYLIP [14]. Reltedness of clone lirries etween pired soils nd medium types were compred using -LIBSHUFF [35], nd rrefction curves, diversity indices, nd linege through time were evluted using DOTUR [35]. Results Soil Physicl nd Chemicl Anlyses Pired Anthrosol nd djcent soil smples from ech site differed mrkedly in soil color, with Anthrosols hving drk, lckish color down to 1 m or deeper, which is due primrily to the presence of BC. Grphitic BC is portion of totl BC (Tle 1) nd, while it does not serve s proxy for the highly heterogeneous BC, it is elevted in the Anthrosols [26]. Adjcent soils hd yellowish to deep ornge color with slightly drker surfce O horizon. Pired smples listed in Tle 1 were chosen sed on visul oservtion of soil horizons, including fctors such s rooting depth nd ioturtion. Adjcent soils t Ht nd LG hd higher percentge of snd nd cly thn the Anthrosols, which contined from two to over six times more silt thn the djcent soils. Anthrosols generlly hd higher ph, higher ction exchnge cpcity (CEC), nd considerly higher totl P nd C contents (the ltter to lesser extent in Acu). Orgnic C nd totl N were similr etween the pired soil smples from ech site. At the Ht nd LG sites, C/N rtios in the Anthrosols were significntly higher thn those of the djcent soils (Tle 1). MPN in SEL nd R2L All of the Anthrosols cultured in oth SEL nd R2L medi hd MPN estimtes within the sme order of mgnitude, regrdless of site or smpling depth, except for the lowest horizons t the Ht nd Acu sites, which were elow the estimted rnge of surfce soil popultions (Fig. 1). Adjcent soils hd popultion estimtes within the sme order of mgnitude s their pired Anthrosols in only three of the seven pired smples (Ht, 0 30 nd cm; LG, 0 10 cm). At ll sites, the estimted numer of culturle orgnisms declined considerly fster with depth in the djcent soils thn in the Anthrosols. In the djcent soils t

5 Microil Communities in Brzilin Anthrosols nd Adjcent Soils 27 Figure 1 MPN (log 10 ) of cteri CFU per grm ODW soil y site, depth, nd soil (lck r Anthrosol, gry r djcent soil) in SEL nd R2L. MPNs were clculted using the MPNES softwre [46]. Letters over ech r indicte stndrd errors (95% confidence intervl) within the sme or different rnge Log10 CFU g -1 ODW soil A d c SEL d Ht LG Acu Soil smpling depths (cm) nd sites Log10 CFU g -1 ODW soil B c R2L c Ht LG Acu Soil smpling depths (cm) nd sites the Ht nd Acu sites, popultion estimtes for the deepest depths were more thn two orders of mgnitude lower thn surfce popultions. Selective Medium Nutrients The ph of soil extrct medi rnged from 7.02 to 7.36 (dt not shown) for ll smples nd ws not considered mjor vrile in these experiments. The ion concentrtion of slts in the commercil R2A medium ws lower thn most elements extrcted from the soil smples used to mke the soil extrct medi (Fig. 2). Exceptions to this were concentrtions of potssium (K) nd P in djcent soil extrcts. Nitrogen (N) concentrtions were lower in SEA compred to R2A (from the contriution of N slts lone) y n verge of 24.7%, with SEA from the Anthrosols generlly more N-deficient thn SEA from djcent soils. Nitrte nitrogen concentrtions were lower in ll SEA medi y more thn n order of mgnitude compred to NO 3 N provided in the slts dded to R2A. Between the pired soil extrcts, the Anthrosols generlly hd lower NH 4 + N nd NO 3 N concentrtions thn the djcent soils; however, N concentrtions in djcent soil smples decresed more rpidlywithsoildepth. Isolte Growth nd Screening Nerly ll cteril colonies formed on R2A pltes developed during the first week of incution for ll sites, soil types, nd depths, while on SEA, colonies continued to form over weeks to months (Fig. 3). Appernce of new isolte colonies y soil type, depth, site, nd medium type nd ll interctions cross time ws fstest on R2A in week 1 for the Anthrosols smpled t shllow depths (p=0.0086) nd on R2A for djcent soil surfce nd susurfce smples (p= nd p=0.0071, respectively). Colony development on SEA from djcent soils occurred over longer period, with greter proportion of colonies forming 2 weeks or more fter inocultion. As with the MPN from liquid cultures, totl colony-forming units (CFU) per grm of soil dropped off fster t deeper depths in the djcent soils thn in the Anthrosols on oth R2A nd SEA (dt not shown). All cteril isoltes grouped into 86 different

6 28 B. O Neill et l. Figure 2 Comprison of nutrient concentrtions in plting medi. Bold dshed lines represent concentrtions in R2A medium. Depths (in centimeters) correspond to different smpling depths s in Fig. 1 (squres from site LG, circles from Acu, tringles from Ht). Open symols with dotted lines re from djcent soils nd filled symols with solid lines re from Anthrosols NO 3 (log mg/l) NH 4 (log mg/l) N (log ppm) P (log ppm) K (log ppm) C (log ppm) Mg (log ppm) Fe (log ppm) Depth (cm) Depth (cm) riotypes from which t lest one representtive ws selected for sequencing. Isolte Sequencing The Anthrosol sequences were rodly distriuted cross mny tx of the cteri, while over 80% of the sequences recovered from djcent soil isoltes grouped to Bcillus or Penicillus (Fig. 4). Comprison of isoltes selected y use of the different medi showed tht, roder distriution of cteri, sed on riotype, ws recovered on SEA, while most isoltes (65%) tken from R2A pltes were Bcillus species. Rrefction curves for the 16S rrna sequences recovered showed the Anthrosols to hve significntly higher numer of unique species mong the 30 isoltes smpled for ech site soil compred to the djcent soils (Fig. 4) (p= 0.05). No such divergence ws oserved when the numer of unique sequences recovered from R2A nd SEA medi were compred. Comprison of these lirries sed on soil nd medium type ws crried out using -LIBSHUFF to mesure genetic distnce in lirry coverge. In this nlysis,

7 Microil Communities in Brzilin Anthrosols nd Adjcent Soils 29 Log CFU/g D1-R2A D1-SEA D2-R2A D2-SEA Week Figure 3 Growth curves sed on colony ppernce during incution t 30 C on R2A nd SEA grouped into weekly oservtions, with mens cross three sites for upper (D1) nd lower (D2) soil depths fitted with exponentil growth curves lirry, C X, is nlyzed for singleton sequences cross the evolutionry distnce contined within the 16S rrna gene nd the chnge in coverge, C XY, is compred to pired lirry. The reciprocl comprison is then mde using the other lirry, C Y, nd the coverge chnge C YX. p vlues derived y compring the lirries from Anthrosol isoltes nd djcent soil isoltes were significntly different cross the sme genetic distnce, showing distinct compositions of isoltes from ech soil (Fig. 4c). In compring isoltes recovered using different medi, the low p vlue for C YX (0.0158) nd higher p vlue for C XY (0.2554) indicted tht the smples from R2A were suset of those isolted on SEA [36], in spite of the ltter hving fewer isoltes. Finlly, in linege through time plots (Fig. 4d), phylogenies s function of time were nlyzed to compre divergent lineges etween the lirries [4]. The greter concvity oserved in the linege from djcent soil isoltes indicted n undnce of closely relted species, s suggested y compring the lirries derived from djcent soils cultured on the R2A medium. A comprtively greter convex curve, s oserved for isoltes recovered from SEA nd from the Anthrosols, indicted higher degree of divergent lineges. Pirwise comprisons of isoltes sed on soil nd medium type showed tht lirries derived from the Anthrosols did not differ from ech other whether they were cultured on SEA or R2A, while djcent soil lirries differed significntly from ech other sed on medium type (Tle 2). Richness estimtes clculted using Cho1 indicted tht the isoltes from SEA prepred from djcent soil hd significntly lower richness, ut tken with SEA isoltes from the Anthrosols, soil extrct medi recovered roder rnge of cteril fmilies thn did the use of R2A to culture isoltes from either soil (Tle 2). Sequencing nd RFLP results suggest tht, roder rnge of tx, identified to the fmily level using RDP II Clssifier [10], ws cultured on Anthrosol SEA thn on SEA from djcent soils. Sequences otined from unique RFLP types showed 19 cteril fmilies mong the isoltes exmined, nd of these, 15 were represented in isoltes from the Anthrosols compred to only eight fmilies represented in the isoltes from the djcent soils, with four fmilies occurring in oth soil types. Furthermore, of the isoltes initilly cultured on SEA, representtives of 16 fmilies were oserved in 49 sequenced 16S rrna gene mplicons from Anthrosols nd only ten fmilies were represented within the 48 sequenced isoltes tken initilly from djcent soil SEA. There ws lso cler pttern in the types of orgnisms predominting in ech soil type. All 14 sequences tht clustered with the Actinocteri cme from Anthrosols nd 11 out of the 14 unique sequences of Proteocteri otined originted from the Anthrosols (Fig. 3). Of 35 unique RFLP types found only from djcent soil isoltes, sequencing reveled 30 elonged to the Firmicutes. Furthermore, sequences from 15 fmilies were derived from susurfce soil smples nd, of these, djcent soils contined representtives from only four fmilies, wheres Anthrosols contined representtives from 13 fmilies. Discussion Pired Anthrosols nd djcent soils used in this study were formed from the sme prent mteril nd experienced the sme previling environmentl conditions. However, culturing, isolting, riotyping, nd sequencing reveled tht the Anthrosols hd higher popultions of culturle cteri to greter soil depths nd contined much higher species richness thn the djcent soils. Out of ll distinct RFLP types generted, oth unique nd those occurring more thn once mong the isoltes, 53% were derived from Anthrosol isoltes, 33% were from djcent soil isoltes, nd 14% shred isoltes from oth soil types (dt not shown). Given tht environmentl conditions were controlled for etween soil types, using soil extrct medium s the selective gent reveled greter fmily-level diversity mong isoltes, especilly in the Anthrosols, thn did stndrd miniml medium (R2A). For six out of seven pired soils cross three sites nd multiple depths, the rtio of cteril fmilies represented to the numer of isoltes recovered ws greter in the Anthrosols thn in the djcent soils. Ten cteril fmilies were represented in the isoltes recovered on R2A, while 15 cteril fmilies were recovered on SEA, despite its lower nutrient contents, slower growth rtes suggesting less ville C, nd greter difficulty isolting nd suculturing from the SEA medi.

8 30 B. O Neill et l. Figure 4 Comprison of soil nd medi y distriution of RF types mong mjor soil cteri types, rrefction curves with 95% confidence intervl error rs, c -LIB- SHUFF nlysis, nd d linege through time plot for 16S rrna gene lirries Soil Medi % RFs % RFs A Bcillus Penicillus Proteocteri Actinocteri Other Anth Adj R2A SEA B Numer of OTUs Oserved C Anth Adj Numer of Sequences Smpled 1.2 Numer of OTUs Oserved R2A SEA Numer of Sequences Smpled Coverge (%) CX p= CXY CY p= CYX Coverge (%) CX p= CXY CY p= CYX Evolutionry Distnce Evolutionry Distnce D Anth Adj 0 R2A SEA Numer of OTUs 10 Numer of OTUs Evolutionry Distnce Evolutionry Distnce R2A is stndrd miniml medium designed to cultivte n rry of slow-growing, heterotrophic orgnisms from mny environments [31], ut it does not reflect the nutrient lnces constrining microil growth in mny soils [33]. In this study, colonies formed more rpidly on R2A medium for ll soil smples (Fig. 3), suggesting tht it fvored growth of distinct group of copiotrophic orgnisms [15]. Of the colonies isolted on R2A, 38% grew exclusively on this medium nd not on the SEA derived from the sme smple. Of these, 84% grouped with the Bcillcee nd Penicillcee, which likely reflects recovered spores rther thn ctively metolizing cells. Of the isoltes from SEA, 19% grew only on their respective SEA nd not on R2A. Of these, less thn hlf were in the

9 Microil Communities in Brzilin Anthrosols nd Adjcent Soils 31 Tle 2 Pirwise comprisons of RF types cross ll sites from two soil types, isolted on two medi, using -LIBSHUFF, Cho1 richness estimte, nd numer of cteril fmilies per numer of isoltes Soil Medium p vlue, using -LIBSHUFF Estimted richness Cho1 (0.05 evolutionry distnce) Adj Anth Bcteril fmilies/isoltes SEA R2A SEA R2A Adj SEA /28 R2A /51 Anth SEA /45 R2A /66 Significnt difference from other vlues for respective ssy Bcillcee nd Penicillcee. Slower cteril growth on SEA nd its selectivity (Fig. 3) my e due to differences in C vilility, sustrte qulity, nd the lck of key nutrients, prticulrly P for djcent soils nd N for oth soil types (Fig. 2), nd this should reflect constrints to microil growth in situ. Adjcent soil SEA micronutrients nd N content decresed significntly with soil depth nd so did cteril popultions in R2L (Fig. 1) nd on R2A (Fig. 3), while higher nutrient contents t deeper depths in the Anthrosols corresponded with higher numers of culturle cteri on ll medi (Fig. 1). These results suggest tht differences in the diversity of cteri recovered from the different medi re not simply due to the lower nutrient contents in the soil extrct medi. Anlysis of PCR-RFLP riotypes nd phylogeny of 16S rrna gene mplicons showed distinct ptterns in lirry composition sed on soil type nd the medium on which colonies were initilly isolted (Fig. 4). Riotypes of isoltes from the Anthrosols were more evenly distriuted etween cteril groups thn isoltes from the djcent soils nd, similrly, SEA medi cptured roder distriution of cteri thn R2A (Fig. 4). Bsed on 16S rrna gene sequences, isolte lirries from the different soils differed significntly oth for species rrity nd coverge (Fig. 4, c), with cler phylogenetic divergence etween isolte lirries from ech soil type (Fig. 4d). These trends were less pronounced when lirries were compred ccording to medium type, suggesting tht selection is sed on medium ws not s strong s the underlying differences sed on soil type. Pirwise comprisons of soil nd medium type cross ll sites showed significnt difference etween cteri selected y R2A versus SEA for djcent soils, ut no similr divergence y medium for the Anthrosols (Tle 2). SEA cptured greter fmily-level diversity, while R2A incresed estimted richness y strongly fvoring growth of unique Firmicutes species, prticulrly for the djcent soils (Fig. 5; Tle 2). The higher culturle cteril diversity in the Anthrosols sed on riotype ptterns nd fmily-level sequence clssifiction nd the higher estimted popultions over greter soil depth suggest ecosystem-level differences in these soil microil communities. Most Proteocteri isoltes nd ll sequenced Actinocteri isoltes were recovered from the Anthrosols nd, of these, most were initilly isolted on SEA from smples tken from the lower horizons (Fig. 5). As soil susystem, the Anthrosols, with higher minerl nutrient contents s well s BC t lower depths (Figs. 1, nd 2), hve n extended re for root growth nd thus incresed rhizosphere interction with microes, leding to lrger volume for physicl nd chemicl interctions, compred to the shllow zone of decomposition nd nutrient resorption in mny tropicl soils [43]. The undnce of grm-positive Firmicutes recovered from surfce soils, especilly in djcent soils on R2A (Figs. 4 nd 5), my represent more ephemerl ecology of orgnisms dpted to rpid growth when nutrients ecome ville [17] nd spore formtion when nutrients re scrce or moisture is limited. A greter diversity of Actinocteri in the BC-rich Anthrosols suggests tht these orgnisms my ply n importnt role in nutrient dynmics in these soils. The presence of BC hs een shown to gretly increse soil CEC [26]. BC prticles lso hve n extremely high surfce re, highly romtic, reclcitrnt structure, nd their surfces undergo decomposition nd oxidtion over time [25]. Actinocteri re physiologiclly dpted to degrde cron-rich, reclcitrnt mterils [1, 27] nd their rnching morphology my llow them to permete the porous structure of BC thus incresing surfce nutrient exchnge. The high numer of unique sequences in isoltes recovered on Anthrosol SEA medi my represent orgnisms distinct to this soil type, s opposed to R2A, which my select for isoltes tht co-occur in oth soil types or re functionlly redundnt. Bsed on similrity to dtse mtches nd ootstrp vlues in constructing multiple trees [12], the screened isoltes yielded five new puttive cteril fmilies. Three of the five puttive new fmilies were recovered from the djcent soil isoltes, two of

10 32 B. O Neill et l. Figure 5 Phylogenetic tree for isolte 16S rrna gene sequences from Anthrosol (Anth) nd djcent (Adj) soils. Ech isolte entry lists the soil type, the depth rnge (in centimeters) of soil smple where it ws found, followed y the medium (SEA or R2A) on which it ws initilly isolted, nd the lternte medium if the isolte could e cultured fter reciprocl plting. Reference sequences re in old with GenBnk ccession numers in prentheses A Anth SEA/R2A Anth 10-40SEA/R2A B. thuringiensis (DQ286359) Anth R2A Anth 0-30 R2A/R2A Anth SEA/R2A B. mycoides (AY373357) Anth 0-30 R2A/SEA Anth SEA/R2A Anth 0-30 R2A Anth 0-30 R2A Anth 0-30 R2A/SEA B. luciferensis (AJ419629) Anth SEA/R2A Anth 0-30 R2A Anth 0-30 R2A uncultured Firmicutes (EF071363) Anth R2A Anth 0-30 SEA/R2A Bcillus sp. (DQ280367) 51 Anth 0-30 SEA/R2A B. clusii (AB201794) Anth 0-30 SEA/R2A Anth 0-30 SEA/R2A Anth 0-30 SEA/R2A Anth 0-30 SEA/R2A B. yunchengensis (EU046268) Anth 0-30 R2A/SEA Anth R2A/SEA Anth R2A/SEA L. sphericus (AB116123) Anth SEA/R2A Anth R2A/SEA B. fusiformis (DQ333300) Anth SEA/R2A B. reuszeri (AB112715) Anth R2A P. pnciterre (AB245385) P. lvei (AB073200) P. mcerns (AB073196) Anth 0-30 R2A Anth SEA/R2A Anth R2A Penicillus sp. (AB089250) Anth R2A P. chinjuensis (AF164345) Anth SEA/R2A Streptomyces sp. (EF012108) Anth R2A/SEA S. griseovriilis (AY654298) Anth SEA Anth 0-10 R2A/SEA Microcterium oxydns (AY509222) Anth 0-30 SEA/R2A Anth 0-10 SEA Tetrpher sp. (AF409018) Anth R2A Anth R2A/SEA Arthrocter sp. (AY452080) Anth 0-10 R2A/SEA Anth SEA/R2A Arthrocter sp. (EU034524) Anth R2A/SEA Anth SEA/R2A Anth SEA/R2A Anth SEA/R2A Anth SEA/R2A Anth SEA/R2A Anth 0-30 R2A/SEA uncultured Pseudomons (EU305565) Anth 0-30 SEA Enterocter sp. (AB114268) uncult. gmm proteocterium (DQ451462) Anth 0-10 SEA/R2A Anth 0-10 SEA/R2A Sphingomons sp. (AB033949) Anth SEA/R2A Anth R2A Anth SEA/R2A Mesorhizoium sp. (AY875973) Anth R2A uncultured cterium (AJ863369) Anth R2A/SEA Brdyrhizoi genosp. (AJ785292) Anth 0-10 SEA/R2A Brdyrhizoium sp. (AF510586) Anth R2A/SEA Gmm Alph Bcillcee Penicillee Streptomycetcee Mycoctericee Microctericee Intrsporngicee Micrococccee Proteocteri which were recovered on R2A nd clustered within the Bcilliles ndonethtclusteredwiththebcteriodetes, which ws initilly recovered on SEA. The two other puttive fmilies cluster within the α-proteocteriles nd oth of these were represented in isoltes recovered from the Anthrosols cultured on R2A. A possile 30 new species were recovered, mostly on SEA medi, lthough verifying this will require sequencing of housekeeping genes [38]. Other culture-independent studies on Amzonin soil microil ecology hve ttriuted vrition in cteril diversity to plnt communities nd lnd use chnge [5, 47], while differences in cteril species richness etween Anthrosols nd their djcent soils hve een demonstrted under similr oveground mngement nd ecologicl conditions [22]. In this study, isoltes tht grew on R2A were similr etween the soil types, ut isoltes tht grew on SEA diverged sed on soil type, suggesting tht

11 Microil Communities in Brzilin Anthrosols nd Adjcent Soils 33 Figure 5 (continued) B B. megterium(eu221388) Adj R2A Adj SEA/R2A B. flexus (EF157301) Adj R2A/SEA Adj 0-30 R2A/SEA Adj 0-10 R2A Adj 0-10 SEA/R2A B. thuringiensis (DQ286359) Adj 0-10 R2A/SEA Adj 0-10 R2A/SEA Adj 0-10 R2A/SEA Adj 0-10 SEA/R2A B. licheniformis (EU256500) Adj 0-30 R2A/SEA Adj HT R2A Adj R2A uncultured Firmicutes (EF071363) Adj AC23-9 SEA/R2A Adj 0-30 SEA/R2A B. soli (AJ542513) Adj SEA/R2A Bcillus sp. (EU072714) Adj R2A/SEA L. sphericus (AB116123) Adj R2A/SEA Adj R2A/SEA B. reuszeri (AB112715) Adj 0-10 R2A/SEA Adj 0-30 R2A/SEA Adj R2A Brevicillus sp. (DQ350828) Adj 0-30 R2A/SEA Adj 0-30 SEA Adj R2A/SEA P. lvei (AB073200) Penicillus sp. (AY839866) Adj R2A Penicillus sp. (AB089250) Adj 0-10 SEA/R2A uncultured cterium (EF2031) Adj R2A/SEA Adj R2A Adj 0-30 R2A/SEA uncultured cterium (EU133463) Adj 0-30 SEA/R2A Brdyrhizoium sp. (AF510586) Adj 0-30 SEA/R2A Burkholdri cepci (AY741332) Flexicter sp. (AY238335) Adj 0-10 SEA/R2A Alph Bet Bcteroidetes cterium (AB362776) Adj SEA/R2A Bcillcee Penicillcee Unknown Proteocteri Flexictercee Crenotrichcee underlying edphic fctors re ffecting microil community composition. Both soils were derived from the sme prent mteril, ut modifiction of the Anthrosols sometime etween 600 nd 2,300 yers BP, tht included incorportion of BC [29], led to greter silt content nd reduced snd percentges in these soils (Tle 1). Furthermore, in spite of the high leching potentil, BC incresed CEC in these Anthrosols [26] nd resulted in incresed ph nd higher concentrtions of P nd C, compred to the djcent soils (Fig. 2; Tle 1). Also, due to its lrge surfce re nd microporous structure, BC itself my serve s n idel hitt for microes [30, 40]. Together with differences in soil texture nd the resulting effects on soil moisture, the distinct sptil heterogeneity in the Anthrosols my lter the microil community composition [49]. The reltive undnce of nutrients nd the high C/N rtio (Tle 1) indicte tht N dynmics in the Anthrosols will likely differ significntly from those in djcent soils. Indeed, BC introduced into soil hs een shown to increse nitrifiction nd iologicl nitrogen fixtion [13, 32]. The presence of BC nd resultnt edphic effects likely chnge the fctors driving competition in the Anthrosol microil communities [11] nd the growth of Proteocteri nd Actinocteri on SEA my indicte tht these groups re physiologiclly ctive nd importnt to the functioning of the Anthrosol soil community. Using environmentlly derived culturing medi, we recovered high numer of geneticlly distinct orgnisms nd showed cler differences in community composition etween the Anthrosols nd their djcent soils. Bcteril popultions in the Anthrosols were significntly higher nd more diverse, in spite of the sme previling climte nd prent mteril etween the pired smples, prticulrly in the susurfce smples. Adjcent soil microil communities contined n undnce of r-selected cteri, chrcteristic of rpid C minerliztion rtes, while the Anthrosols were rich in K-selected cteri typicl for more stle environments, with slower rtes of C turnover [16]. The presence of reclcitrnt BC in the Anthrosols my crete chemicl conditions tht foster stle popultions of unique

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