Biogeochemistry D O I 10.1007/S10533-006-9065-Z

ORIGINAL PAPER

Increases in soil respiration following labile carbon additions linked to rapid shifts in soil microbial community composition

Cory C. Cleveland • Diana R. Nemergnt Steven K. Schmidt • Alan R. Townsend

Received: 16 June 2006 / Accepted: 10 November 2006 © Springer Science+Business Media B.V. 2006

Abstract Organic matter decomposition and soil determined by constructing clone libraries of

CO 2 efflux are both mediated by soil microorgan­ small-subunit ribosomal RNA genes (SSU rRNA) isms, but the potential effects of temporal varia­ extracted from the soil at the end of the incubation tions in microbial community composition are not experiment. In contrast to the subtle effects of considered in most analytical models of these adding water alone, additions of DOM caused a two important processes. However, inconsistent rapid and large increase in soil CO2 flux. DOM- relationships between rates of heterotrophic soil stimulated CO2 fluxes also coincided with pro­ respiration and abiotic factors, including temper­ found shifts in the abundance of certain members ature and moisture, suggest that microbial com­ of the soil microbial community. Our results munity composition may be an important regulator suggest that natural DOM inputs may drive high of soil organic matter (SOM) decomposition and rates of soil respiration by stimulating an opportu­

CO 2 efflux. We performed a short-term (12-h) nistic subset of the soil bacterial community, laboratory incubation experiment using tropical particularly members of the Gammaproteobacte- rain forest soil amended with either water (as a ria and Firmicutes groups. Our experiment indi­ control) or dissolved organic matter (DOM) cates that variations in microbial community leached from native plant litter, and analyzed the composition may influence SOM decomposition effects of the treatments on soil respiration and and soil respiration rates, and emphasizes the need microbial community composition. The latter was for in situ studies of how natural variations in microbial community composition regulate soil biogeochemical processes. C. C. Cleveland(E3) ■ D. R. Nemergut ■ A. R. Townsend INST AAR: An Earth and Environmental Sciences Keywords Carbon cycle ■ Decomposition ■ Institute, University of Colorado, 450 UCB/1560 30th Dissolved organic matter ■ DOM ■ Microbial Street, Boulder, CO 80303, USA community composition ■ Organic matter ■ Soil e-mail: [email protected] respiration D. R. Nemergut Environmental Studies Program, University of Colorado, 397 UCB, Boulder, CO, USA Introduction

S. K. Schmidt ■ A. R. Townsend Department of Ecology and Evolutionary Biology, Organic matter decomposition is a fundamental University of Colorado, 334 UCB, Boulder, CO, USA process, regulating rates of net carbon (C)

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Storage and nutrient cycling in terrestrial eco­ a single site, rhizosphere microbial community systems. Decades of research have informed the composition and activity vary by plant species development of ecosystem models that describe and across seasons (Grayston et al. 1998; Bardg- decomposition and heterotrophic soil respiration ett et al. 1999; Wardle et al. 2004; Carney rates primarily as functions of abiotic environ­ and Matson 2005; Schmidt et al. 2007). Second, mental factors including temperature, moisture, manipulative experiments have shown that and organic matter substrate quantity and specialized organisms respond to certain C quality (Meentemeyer 1978; Swift et al. 1979; compounds. Padmanabhan et al. (2003) found Scott-Denton et al. 2003; Davidson and Janssens that specific and unique soil bacterial assem­ 2006; Scott-Denton et al. 2006). However, the blages were responsible for the decomposition of potential effects of temporal variations in micro­ individual soil organic compounds, including bial community structure on these processes are glucose, naphthalene, phenol, and caffeine. seldom explicitly acknowledged (but see Fang Finally, the types of microorganisms within a et al. 2005). Instead, most conceptual and site that respond to C inputs vary through time, empirical decomposition models treat the micro­ suggesting that temporal shifts in community bial community as a “black box” in which structure may directly affect soil respiration rates microorganisms act as passive catalysts of following C inputs. For example, in a subalpine decomposition reactions of substrates whose forest soil, C inputs consistently stimulated rates vary as functions of temperature and members of the Betaproteobacteria, but the moisture (Raich and Schlesinger 1992; Lloyd specific types of soil Betaproteobacteria that and Taylor 1994; Parton et al. 1994). responded in summer were genetically and Simple soil respiration-climate relationships physiologically distinct from those responding are convenient for models attempting to describe in winter (Monson et al. 2006). ecosystem CO 2 fluxes, but many studies show These studies and others support proposed contradictory responses of soil respiration to links between microbial community composition variations in climate (Davidson and Janssens and ecosystem function (Adams and Wall 2000; 2006). Soil moisture correlates with soil respira­ Hooper et al. 2000; Wardle et al. 2004). Unfor­ tion rates in some sites (e.g., Buchmann 2000; tunately, relatively little is known about the types Schwendenman et al. 2003), but only weakly of microorganisms that decompose native soil predicts rates of soil respiration in others (Scott- organic matter (SOM), or how variations in Denton et al. 2003; Cleveland and Townsend microbial activity in response to labile C inputs 2006; Monson et al. 2006). The inability of are related to soil microbial community compo­ climate to adequately explain variation in soil sition. Here, we asked a simple question: in an respiration rates suggests that soil CO2 fluxes are ecosystem where high rates of soil respiration are regulated by a more complex set of variables and positively related to seasonal increases in C interactions. availability (Cleveland and Townsend 2006),

For example, heterotrophic CO2 fluxes also could seasonal increases in soil respiration be depend on the availability of soluble, labile C related to variations in microbial community sources, and microbial respiration rates are composition? To begin to address this question, tightly linked to the chemistry and amount of we performed a soil incubation experiment using organic matter entering the soil (Schlesinger and soil collected from a mature, lowland tropical rain Andrews 2000; Wardle et al. 2004). In addition, forest. Previous research from the study site three emerging lines of evidence suggest that revealed a strong, positive relationship between short- and long-term variations in soil microbial labile C availability and soil respiration rates community composition (e.g., Schmidt et al. (Cleveland and Townsend 2006), and we hypoth­ 2007) could also influence soil respiration rates. esized that dissolved organic matter (DOM)- First, microbial community structure varies sig­ stimulated increases in soil respiration coincide nificantly within soil types—both spatially and with rapid, measurable shifts in soil microbial temporally—^with research indicating that within community composition.

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Materials And Methods litter traps in the dry season (litter harvested at 2-week intervals throughout the year; Cleveland Study sites and sampling regime and Townsend 2006) were extracted in 1 L of de-ionized water for 12 h at 22°C, pre-filtered Soil and litter samples were collected from a to 0.45 fim and sterile filtered to 0.22 fim. The primary tropical rainforest on the Osa Peninsula C concentration of the resulting leachate in southwestern Costa Rica (see Cleveland et al. (650 mg/L) was measured using a Shimadzu 2006 for complete site description). Soils at the TOC 5050A total organic carbon analyzer site are highly weathered, nutrient poor Ultisols. (Shimadzu Corporation, Kyoto, Japan). Sterility The site lies within the tropical wet lowland forest of the leachate was confirmed using Biolog bioclimate, with an average annual temperature microtiter plates (Biolog Inc., Hayward, CA, of 26.5°C (Holdridge et al. 1971). Average annual USA); no color development was observed after rainfall exceeds 5,000 mm (Cleveland and Town­ 24, 48 or 72 h, indicating that the leachate was send 2006), but like most tropical rainforests, this free of all viable bacteria. Approximately 25 g area experiences a dry season (from January of field-moist soil (-15 g dry weight) were to March on the Osa Peninsula). During the placed in 1 L glass vessels and pre-incubated 3-month dry season, more than half of the annual at 26°C (±1°C) for 24 h in a Precision 815 low- litter falls on the forest floor (>500 g C m^; temperature incubator (Precision Scientific, Cleveland and Townsend 2006), providing a large Winchester, VA, USA). pool of soluble, highly decomposable C (e.g., Don After pre-incubation and equilibration at 26°C, and Kalbitz 2005) that may fuel high rates of soil all vessels were aerated and samples(N = 10 respiration during the early rainy season (Cleve­ replicates per treatment) received -5 mL doses land and Townsend 2006). In this experiment, our(i.e., an amount to bring each soil sample to -50% objective was to simulate a DOM pulse leached of water holding capacity) of DOM (-225 fig from litter layer during a low-intensity rain­ DOM-C g“^ soil; -hDOM). T o separate water (soil storm—an event that occurs almost daily in this wet-up) versus C-addition effects on microbial site during the early rainy season (Cleveland and community activity and composition, we also Townsend 2006). amended a parallel set of samples with -5 mL

We collected 8 x 10 cm surface soil cores from of de-ionized water(N = 10 replicates; -HH2 O). each of ten 25 m^ plots in the dry season using a Following treatment additions, glass vessels were hand corer (see Cleveland et al. 2006 for soil immediately sealed with caps equipped with physical and chemical characteristics). Immedi­ rubber septa for gas sampling, incubated at 26°C ately after collection, soil samples were trans­ in darkness for 12 h, and sampled for CO2 at 2, 4, ported on ice in an insulated cooler to the 7, 9, and 12 h using glass gas-tight syringes. laboratory (within 72 h of collection), and stored Sample C O 2 concentrations were determined at at ~4°C until analysis. Soil samples were gently each time point using a Shimadzu GC-14 gas hand-homogenized (to 4 mm) to remove roots chromatograph equipped with a thermal conduc­ and other organic debris. Following homogeniza­ tivity detector. tion, samples were split into paired experimental samples. All incubation experiments were initi­ DNA extraction and analysis ated within 96 h of soil sampling to avoid artifacts incurred during long-term storage. Our strategy was to sample soil microbial com­ munity composition when soil respiration Soil incubation experiment achieved maximum rates. Previous experiments showed that following C additions to these soils, To simulate an episodic DOM leaching event, microbial respiration rates were highest after we added soluble organic material leached from -12 h of incubation at 26°C (Cleveland et al. freshly fallen litter samples to soil. About 100 g 2002). Therefore, after the 12-h headspace CO 2 of mixed, air-dried plant litter obtained from sampling was complete, -5 g of soil from each

^ Springer Biogeochemistry microcosm were removed to sterile conical tubes (20 min.) and re-suspended in 50 mL Tris-HCl and preserved at -80°C until DNA extraction. pH 8.5. Inserted 16S rRNA genes were PCR Soil DNA was extracted from soil samples amplified from the plasmids using the primers using a modification of the protocol described by M13F and M13R (Invitrogen Inc.), and purified More et al. (1994). Briefly, 0.5 g of soil from each 16S rRNA genes were sequenced with the T7 of the +H 2 O and +DOM samples were agitated in promoter primer and the M13-9 primer using the 1 mL phosphate extraction buffer using a bead BigDye Terminator Cycle Sequencing kit Version beater and centrifuged at 14,000g for 10 min. The 3.0 (PE Biosystems, Foster City, CA, USA) resulting supernatant was removed to a fresh tube according to the manufacturer’s directions. and incubated on ice for 5 min with 200fih of Sequencing products were analyzed at the Iowa 7.5 M ammonium acetate, centrifuged at 14,000g State University DNA Sequencing Facility. for 3 min, and the supernatant transferred to a fresh tube. Samples were then extracted with an Data analysis equal volume of phenol : chloroform : isoamyl alcohol (25:24:1), DNA was precipitated with All soil respiration data were tested for homo- 200 fih of isopropanol overnight at -20°C and scedasticity (Levene’s test for equal variances), washed with 1 mL of 70% ethanol. Following normality, and skewedness (SPSS, Chicago, IE, extraction, the individually extracted DNA sam­USA). Significant differences in maximum respi­ ples from each treatment were pooled and puri­ ration rates following DOM additions in the fied over Sepharose 4B packed columns (Sigma, incubation experiment were tested with a St. Louis, MO, USA) as described in Jackson paired-samples t-test (a = 0.05; Snedecor and et al. (1997). Cochran 1989). DNA sequences were edited in Sequencher 4.1 PCR, cloning, and sequencing (Gene Codes Co., Ann Arbor, MI, USA) and subjected to BLAST searches (Altschul et al. Approximately 30 ng of DNA were amplified 1990). The 16S rRNA gene sequences were also with universal bacterial 16S rDNA primers [8f subjected to chimera check in RDP (Cole et al. and 1492r (Lane 1991)]. Each reaction included 2003) and Bellerophon (Huber et al. 2004). These 400 uM of each primer, 200 fiM of each dNTP, sequences were aligned in Dr. Phil Hugenholtz’s 1.25 U of Taq DNA polymerase (Promega, 16S rRNA ARB database (http://rdp8.cme.msu. Madison, WI, USA) in Taq DNA polymerase edu/htmPalignments.html) in ARB (Ludwig et al. buffer [2.5 mM MgCli (Promega)j. After an 2004). Phylogenetic affiliations were assigned initial denaturation step at 94°C (1 min), PCR based on both the ARB alignment and nearest was performed using 30 cycles at 94°C for 1 min, BLAST matches. Operational taxonomic unit 58 ± 5°C for 30 s and 72°C for 2.5 min with a (OTU) designations were assigned using DO- terminal 10 min extension at 72°C. Resulting TUR (Schloss and Handelsman 2005) with a PCR products were gel purified using QIAquick distance-based phylogenetic tree constructed in gel purification columns (Qiagen, Valencia, CA, ARB. Lineage-per-time plots were also con­ USA), ligated into the vector TOPO 2.1 (Invitro- structed in DOTUR (Schloss and Handelsman gen Inc., Carlsbad, CA, USA) and transformed 2005). Detailed phylogenies of the Gammaprote- into Escherichia coli following the manufacturer’s obacteria and the Firmicutes were constructed by instructions. aligning close relatives from the ARB database Transformants were inoculated into 96-well and BLAST matches with our sequences in ARB. deep-dish plates containing 1.5 mL Luria broth Alignments were used to generate a neighbor- (1% NaCl, 1% tryptone, 0.5% yeast extract) and joining phylogenetic tree in PAUP (Swofford 50 mg mL“^ ampicillin. Cultures were agitated at 2001) using the distance optimality criterion. 200g for 14 h at 37°C, plasmid DNA was All trees were subjected to bootstrap analyses extracted and purified (Sambrook and Russell (1,000 replicates) using both the parsimony and 2001), and pelleted DNA samples were air-dried neighbor-joining optimality criterion.

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Results soil respiration in the -HH2 O samples did not differ significantly over the 12-h incubation (Fig. lA ). Soil respiration rates Soil microbial community composition Dissolved organic matter additions (3,375fig C as DOM) rapidly stimulated respiration in the soil We analyzed a total of 102 sequences from the microcosms (Fig. 1). After the 12-h incubation, -HH2 O (N = 46) and -hDOM samples (N = 56), soil respiration rates in the -hDOM samples and there were some notable similarities in were 35.1 ± 1.7 fig CO 2 -C g“^ hr^, versus only community composition between the two treat­

9.1 ± 1.2 fig C O 2 -C g“^ h“^ in the -HH2 O samples ments. Overall, broad-scale (i.e., division- and (Fig. lA). Twelve hours after the DOM addition, sub-division-level) diversity was similar in both the -hDOM samples had respired -100% more sets of samples, with sequences within nine and

CO 2 than the control soil samples (2,981 ± 245fig ten major bacterial clades represented in the

CO 2 -C vs. 1,503 ± 80 fig C O 2 -C in -hDOM and -HH2 O and -hDOM samples, respectively (Fig. 2).

-HH2 O samples, respectively; Fig. IB). In contrast, Sequences related to several of these clades the soil wet-up (water only) had little effect on were found in both the -HH2 O and -hDOM soil soil respiration rates. Immediately following samples, including the Acidobacteria, Firmicutes, water additions, soil respiration rates in -HH2 O Alphaproteobacteria, Betaproteobacteria, Delta- samples increased slightly (Fig. lA ), but rates of proteobacteria, Verrucomicrobia, and the Plan- ctomycetes. However, more detailed comparisons of the communities revealed that, in spite of the similarities between the two treatments at broad­ 40 er phylogenetic levels, the types of organisms 35 within the -HH2 O and -hDOM communities were very different. For example, we compared the c o 25 actual number of unique sequences (>3% 16S 20 rRNA gene sequence difference) in the two {/}Q. a> CC communities over various genetic distances with the theoretical maximum number of possible unique sequences (i.e., if there was no overlap in the two communities), and the theoretical minimum number of possible unique sequences 3500 (i.e., if each sequence present in one community 3000 O was also present in the other community; Fig. 3). C\i 2500 Results indicated that at genetic distances of less

2000 than 10%, the -HH2 O and -hDOM communities were distinct from each other. 1500 Lineage per time (LPT) curves, in which the Q. {/} 1000 <1> number of unique microbial phylotypes is plotted o 500 as a function of genetic distance, provide an index of the amount of phylogenetic diversity within a 0 2 4 6 8 1 0 12 1 4 community. LPT plots for these communities also T im e (h) revealed profound differences between the 2 -HHO

2 Fig. 1 (A) Soil respiration rates; and (B) total CO 2 and -hDOM soils. The -HHO curves showed a respired in soil incubations following water(open circles) gradual decline, suggesting that phylogenetic or dissolved organic matter(DOM', open squares) addi­ diversity was relatively high. In contrast, in the tions (3,375 /ig C as DOM ) to tropical rain forest soil. Soil -hDOM samples, LPT curves showed an initial, respiration rates are expressed on a soil mass(dry -weight) basis. Values represent the mean of ten replicates per relatively steep decline, indicating that many of treatment, anderror bars are ±1 SE the individual phylotypes in the -hDOM samples

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Fig. 2 Relative - Verrucomicrobia Chiorofiexi ^Deitaproteobacteria/—Candidate Division TM7 abundance of 16S rRN A A . Verrucomicrobia Pianctomycetes Nitrospira Betaproteobacteria Pianctomycetes gene phylotypes in (A) rDeltaproteobacterii iacteroidetes -Alphaproteobacteria Acidobacteria Acidobacteria +H 2 O; and(B) +DOM samples. Colors represent division or sub-division- level clades (Proteobacteria) and the black lines represent unique phylotypes (i.e., >3% different from all other sequences)

- Firmicutes - Gammaproteobacteria - Alphaproteobacteria - B e t a p r o t e o b a c t e r ia Firmicutes

100 but our results do show some clear and important • combined data 90 differences. For example, perhaps the most strik­ ' theoretical maximum 80 ‘ theoretical minimum ing difference between -hDOM and control soil

70 communities was the increase in abundance of non-Acidobacteria sequences in the -hDOM soil. 60 CO D Sequences related to the Acidobacteria clade H 50 0 were numerically dominant in both the -HH2 O and 40 -hDOM samples, but they represented nearly 60% 30 of the sequences in the control library and only 20 about 30% in the -hDOM library (Fig. 2). In 10 addition, in the control samples, non-Acidobac- 0 terial sequences included members of nine major 0.1 0.2 0.3 Genetic Distance clades, and the frequencies of individuals within those groups were fairly equally represented; no Fig. 3 Estimated number of unique phylotypes in the single non-Acidobacterial clade represented more combined (+DOM and +H2 O) libraries (circles), com­ than 11% of the sequences, and most individual pared to the theoretical minimum number of phylotypes (i.e., if all sequences in one library were found in the other clades comprised <4% of the sequences. In library; triangles) and the theoretical maximum number of contrast, in the -hDOM samples, non-Acidobac- phylotypes (i.e., if none of the sequences present in one terial sequences were dominated by individuals library were found in the other library;squares). This plot from the Gammaproteobacteria and Firmicutes indicates that although there was some division-level groups, which comprised 34 and 16% of the sequence overlap in the +DOM and +H2 O libraries, the

sequences in the +H2 O and +DOM libraries were very -hDOM sequences, respectively. In the -HH2 O different samples, phylotypes from the Firmicutes group were present in small numbers (-4%), but the sequences were not closely related to the Firmi- were closely related, and that the depth of cute sequences in the -HH2 O samples (data not phylogenetic diversity in the -hDOM samples shown). Sequences related to the Gammaproteo­ was lower than in the -HH2 O samples (data not bacteria were not detected in the -HH2 O samples. shown). Our sampling and analysis of microbial com­ munity composition in the soil samples was not Discussion exhaustive, therefore it is difficult to make defin­ itive statements about the abundance of specific Soil moisture content can limit decomposition bacterial lineages or overall microbial diversity. and soil respiration rates (e.g., Housman et al.

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2006; Potts et al. 2006). However, in this tropical solution concentrations (Schwendenmann and rain forest site, soil moisture (to 10 cm depth) Veldkamp 2005), it did represent realistic con­ remains relatively constant throughout the year, centrations of DOM through the litter layer (Neff even during the 3-month dry season when pre­ and Asner 2001; Cleveland et al. 2004). Previous cipitation is <100 mm/month (Cleveland and research at this site showed that as much as 50% Townsend 2006). In this experiment, water-only of dry season litter C is susceptible to leaching in additions did not significantly enhance rates of the first 60 days of the rainy season (Cleveland soil respiration (Fig. la). The lack of a strong et al. 2006). If so, a typical daily 10 mm rainfall

CO 2 response to the soil wet-up indicates that event in the early rainy season and subsequent even though soil samples were collected in the dry leaching of the dry season litter pool season, soil moisture was not limiting to hetero­ (250 mg C m“^) could generate a pulse of DOM trophic respiration at that time. This result is of -200 mg to the soil surface. The increase in consistent with previous data from this site soil respiration that we observed following addi­ showing that soil moisture does not correlate tions of native, leached DOM suggests that strongly with rates of soil respiration or other soil episodic DOM fluxes in situ may also rapidly processes (Cleveland and Townsend 2006). stimulate high rates of soil respiration. Although seasonal increases in rainfall do not Rapid increases in microbial activity following drive significant variation in soil moisture, they do pulses of labile C inputs are common, but do such facilitate the movement of DOM from litter to increases represent a community-wide response, soil, driving significant dry-to-wet season in­ or are they accomplished via shifts in the abun­ creases in soil respiration (Cleveland et al. 2006; dance of certain microbial taxa? Our results Cleveland and Townsend 2006). In this experi­ provide compelling evidence that increases in soil ment, microbial respiration responded rapidly to respiration following C inputs may be dominated dissolved organic C additions in the-hDOM by a subset of soil microbes, with DOM inputs samples (Fig. 1). Many other studies showing causing rapid shifts in soil bacterial community rapid microbial responses to labile C additions composition. In the -HH2 O samples, as is common have added relatively high concentrations of in many soils, the Acidobacteria was the numer­ simple, pure, C-rich compounds that stimulate ically dominant group, but the abundances of microbial activity, but that may not necessarily be individuals within other division-level groups were analogous to native organic material that is relatively evenly distributed. However, at the peak present in natural ecosystems. Thus, while the of soil respiration, microbial community structure high rates of microbial respiration we observed in in the -hDOM soil was markedly different. In response to C additions (in general) are not particular, C additions appeared to strongly stim­ surprising, the rapid response immediately fol­ ulate organisms within the Gammaproteobacteria lowing additions of relatively dilute, litter-leached and Firmicutes clades. native DOM is noteworthy for several reasons. The dominance of organisms within the Gam­ First, leached DOM is a complex, heteroge­ maproteobacteria and Firmicutes groups, and neous suite of C compounds that vary in their relative increase in abundance in response complexity and decomposability (Neff and Asner to C additions, suggests that members of these 2001; Cleveland et al. 2004), yet it still stimulated groups quickly responded to the additions of rapid rates of microbial respiration in our incu­ organic material added in the-h DOM soil, bation. In addition, previous in situ DOM leading to rapid increases in soil respiration. additions elicited similarly high-respiration rates, Detailed phylogenetic analyses of the Gamma­ suggesting that rapid respiratory responses to proteobacteria and Firmicutes sequences re­ episodic DOM inputs may be important in vealed that they are related to the order regulating ecosystem function in this site (Cleve­ Enterobacteriales and the genus Bacillus, respec­ land et al. 2002). Next, while the concentration of tively (Figs. 4, 5), groups that include an array of the DOM added here (-650 mg L“^) was higher both pathogenic and symbiotic bacteria. Inter­ than typical tropical rain forest throughfall or soil estingly, members of these orders have been

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^ f— Shigella flexnerl Lactobacillus agilis I * Escherichia coll ■[j— — Enterococcus faecalis Salmonella typhlmurlum T » -----I Streptococcus

L, SalmSalmonella enterica I— Bacillus circulans ^.Klebsiella pneumoniae I— Bacillus megaterlum P solubilizer Klebsiella CBNU-34-2 P solubilizer

■I _|L PantoePantoea agglomerans P solubilizer 1Bacillus firmus P solublllzer L EnterobaEnterobacter aerogenes P solubilizer — Saccharococcus thermophllus Kluyvera Intermedlus P solubilizer Fllobaclllus

Gracilibacillus dipsosaurl Graclllbaclllus halotolerans

Pasteurellales Pontlbaclllus I Alteromonadales Lentlbaclllus

Legionellales Oceanobaclllus • Exiguobacterlum Alteromonadales

Paenlbaclllus Methylococcales ______Geobacillus

^ — 0.01 substitutions/site Urelbaclllus Ureaplasm a Fig. 4 Neighbor-joining phylogenetic tree showing repre­ sentative sequences from the clones related to the M ycoplasm a Enterobacteriales in the +DOM samples. Rectangles represent sequences from this study, and the size of the 0.01 substitutions/site rectangle symbolizes the number of sequences obtained. Reference taxa were selected based on nearest BLAST matches. Tree is rooted with Rhodobaeter sphaeroides (X53853), Bartonella baeilliformis (X60042) and Rhizo- bium hainanensis (U71078).Asterisk indicates a parsimony soluble C in soil is strongly phosphorus (P) and distance bootstrap (1,000 replicates) value of 80 or limited (Cleveland et al. 2002; Cleveland and higher. Accession numbers: Shigella flexnerl (X80679), Townsend 2006). While by no means definitive, Eseheriehia eoli (X80724), Salmonella typhlmurlum the presence of relatives of known P-solubilizing (X80681),Salmonella enterlea (AL627282), Klebsiella pneumoniae (X87276), Klebsiella sp. CBNU-34-2 organisms in the -hDOM samples suggests an (AY335553), Pantoea agglomerans (AY335552), Enterob- interesting hypothesis: that the ability to solubi­ aeter aerogenes (AY335552), Kluyvera Intermedlus lize P may give the Enterobacteriales and the (AF297470), Vibrionales [Vibrio natrlegens (X74714), Bacillus a competitive advantage in these P-poor Vibrio eholerae (X74696), Photobaeterlum hlstamlnum (D25308)], Pasteurellales [Alteromonas maeleodll soils, allowing them to rapidly grow in response (X82145), Chromohalobaeter marlsmortul (X87221)], Leg­ to episodic C inputs. ionellales [Eeglonella gormanll (Z32639), Eeglonella pneu­ To our knowledge, our data are among the first mophila (M59157)], Alteromonadales [Franelsella to show that increases in soil respiration following tularensls (L26086), Thlomlerosplra erunogena (L40810)], Methylococcales [Methyloeoeeus eapsulatus (M29023), native DOM additions to soils correspond with a Methyloeoeeus sp. (X72769)] rapid shift in microbial community composition. Yet, rapid shifts in microbial composition in response to changing environmental conditions identified as potential phosphate solubilizers, and/or resources have been observed by microbial capable of accessing calcium-, iron- and alumi­ ecologists for some time. More than 75 years ago, num-bound phosphate minerals (Rodriguez and Winogradsky (1925) observed increases in zymog- Fraga 1999; Chung et al. 2005; Souchie et al. enous organisms following substrate additions, 2006). This is noteworthy given previous work at with a return to more “stable,” slow growing this site indicating that the decomposition of communities following substrate depletion. More

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■^Fig. 5 Neighbor-joining phylogenetic tree showing repre­ groups) may be strongly regulated by C availability sentative sequences from the clones related to the genus (Fierer et al. 2007). However, our results also Bacillus in the +DOM samples. Rectangles represent suggest that microbial community structure and sequences from this study, and the size of the rectangle symbolizes the number of sequences obtained. Reference composition may vary rapidly in response to taxa were selected based on nearest BLAST matches. Tree short-term changes in C availability. In other is rooted with Trichodesmium sp. (X70767), Nostoc words, while low-soil C availability (on average) punctiforme (AF027655) and Gloeothece membranacea may indeed favor oligotrophic organisms, natural (X78680).Asterisk indicates a parsimony and distance bootstrap (1,000 replicates) value of 80 or higher. Acces­ and frequent increases in C availability in many sion numbers:Laetobaeillus agilis (M58803),Enterococcus ecosystems may promote short-term variations in faecalis (AE016830), Streptococcus [Streptococcus pneu­ community composition, and favor the temporary moniae (X58312), Streptococcus macedonicus (Z94012)], dominance of copiotrophic organisms. Bacillus circulans (D78312), Bacillus megaterlum (D16273), Bacillus firmus (D16268), Saccharococcus ther- The increased abundance of Gammaproteo­ mophilus (L09227), Filobacillus [Filobacillus salipiscarius bacteria and Firmicutes sequences (Fig. 2) fol­ (AB194046),Filobacillus milensis (AJ238042)], Graciliba­ lowing DOM additions suggests that members of cillus dipsosaurl (AB101591), Gracilibacillus halotolerans these two divisions may be largely responsible for (AF036922), Pontibacillus [Pontibacillus chungwhensis (AY603362), Pontibacillus marinus (AY603977)], Lenti- the observed increases in soil respiration in our bacillus [Fentibacillus kapialis (AY667493), Fentibacillus incubation experiment, contributing to a growing salarius (AB231905)], Oceanobacillus [Oceanobaclllus body of research showing that members of the chironomi (DQ468656) Oceanobacillus profundus these clades may dominate many important bio­ (DQ386635)], Exiguobacterium[Exiguobacterlum auran- tiacum (X70316), Exiguobacterlum sp. (X86064)], Paeni- geochemical processes in natural ecosystems. The bacillus [Paenibacillus validus (M77489), Paenibacillus importance of the Gammaproteobactria, in par­ azotofxans (D78318)], Geobacillus[Geobacillus thermo- ticular, in soil C cycling has also been shown in glucosidasius (AJ781265)], Ureibacillus [Ureibacillus other studies. Padamanbhan et al. (2003) showed koreensis (DQ348072), Ureibacillus rudaensis (DQ348071)], Ureaplasma [Ureaplasma canigenitalium that phylotypes within the Gammaproteobacteria (D78648),Ureaplasma parvum (L08642)], Mycoplasma clade were responsible for the decomposition of [Mycoplasma capricolum (U26046), Mycoplasma mycoides pulses of suite of organic substrates including (U26044)] naphthalene, caffeine, and glucose. A molecular characterization of phenol-degrading bacteria recently, Fierer et al. (2007) suggested an ecolog­ also showed that close relatives of both the ical classification of bacteria that proposed that Gammaproteobacteria and the Firmicutes were the abundance of copiotrophic and oligotrophic important phenolic compound mineralizers in soil bacteria in soil is regulated by the abundance of (Abd-El-Haleem et al. 2002). These studies, labile C substrates, and that C decomposition among others, demonstrate that members of the rates (which include heterotrophic soil respira­ Gammaproteobacteria and Firmicutes divisions tion) vary directly with copiotrophic bacterial are particularly adept at responding to increases abundance. For example, in low C environments, in labile soil C, and that in systems where natural oligotrophic groups like the Acidobacteria were pulses of diverse C compounds are common, most dominant, but in high C soils, copiotrophic these groups may be disproportionately impor­ groups like the Proteobacteria, and Bacteroidetes tant to the decomposition (and hence respiration) increased in relative abundance (Fierer et al. of labile C compounds. 2007). In our experiment, we observed a similar Traditionally, ecosystem models have focused pattern. DOM additions caused a relative on the role of abiotic factors in regulating organic decrease in Acidobacterial sequences, and matter decomposition rates. However, such sim­ relative increases in Gammaproteobacteria and plifications are often inadequate for explaining Firmicutes groups. Our results support a model in observed variations in soil respiration (Davidson which abundant bacterial phyla may be ecologi­ and Janssens 2006; Monson et al. 2006; Sacks et al. cally classified according to their physiology, and 2006). Our results provide evidence of strong links in which the abundance of these ecologically between variations in both microbial community defined groups (i.e., copiotrophic and ohgotrophic composition and soil respiration, and suggest that

^ Springer Biogeochemistry a complete understanding of decomposition Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ dynamics and soil respiration (and perhaps other (1990) Basic local alignment search tool. J Mol Biol 215:403-410 soil biogeochemical processes) in natural ecosys­ Bardgett RD, Lovell RD, Hobbs PJ, Jarvis SC (1999) tems may require a more thorough understanding Seasonal changes in soil microbial communities along of temporal shifts in microbial community com­ a fertility gradient of temperate grasslands. Soil Biol position. While results from a short-term labora­ Biochem 31:1021-1030 Borneman J, Triplett EW (1997) Molecular microbial tory experiment with limited sample numbers may diversity in soils from eastern amazonia: Evidence for not accurately describe microbial dynamics in situ, unusual microorganims and microbial population they do provide both justification and a frame­ shifts associated with deforestation. AppI Environ work for further research investigating links Microbiol 63:2647-2653 Buchmann N (2000) Biotic and abiotic factors controlling between soil microbial community structure and soil respiration rates inPicea abies stands. Soil Biol function in the natural environment. Many forces, Biochem 32:1625-1635 including land use change (Bornemann and Trip­Carney KM, Matson PA (2005) Plant communities, soil lett 1997; Nusslein and Tiedje 1999), variations in microorganisms, and soil carbon cycling: Does alter­ ing the world belowground matter to ecosystem nutrient fertility (Bardgett et al. 1999), and functioning? Ecosystems 8:928-940 climate variability (Monson et al. 2006), have Chung H, Park M, Madhaiyan M, Seshadri S, Song J, Cho been shown to drive either short-term variations H, Sa T (2005) Isolation and characterization of or perhaps long-term or permanent changes in phosphate solubilizing bacteria from the rhizosphere of crop plants of Korea. Soil Biol Biochem 37:1970- microbial community composition. Understand­ 1974 ing how natural and disturbance-driven processes Cleveland CC, Townsend AR, Schmidt SK (2002) Phos­ affect microbial community dynamics, and how phorus limitation of microbial processes in moist these, in turn, interact with abiotic factors to tropical forests: Evidence from short-term laboratory incubations and field experiments. Ecosystems 5:680- regulate soil biogeochemical processes, may be 691 important in generating more accurate predictions Cleveland CC, Neff JC, Townsend AR, Hood E (2004) of the response of terrestrial ecosystems to envi­ Composition, dynamics and fate of leached dissolved ronmental change. organic matter in terrestrial ecosystems: results from a decomposition experiment. Ecosystems 7:275-285 Cleveland CC, Reed SC, Townsend AR (2006) Nutrient Acknowledgments We thank H. and M. Michaud of the regulation of organic matter decomposition in a Drake Bay Wilderness Camp for providing field access and tropical rain forest. Ecology 87:492-503 logistical support in Costa Rica, and Francisco Campos Cleveland CC, Townsend AR (2006) Nutrient additions to and the Organizacion para Estudios Tropicales (OBT) and a tropical rain forest drive substantial soil carbon the Ministerio de Ambiente y Energia (MINAE) in Costa dioxide losses to the atmosphere. Proc Natl Acad Sci Rica for assisting with research permits and logistics. We USA 103:10316-10321 are grateful to R. Ley for valuable discussions and insight Cole JR, Chai B, Marsh TL, Farris RJ, Wang Q, Kulan during the experimental design and data analysis, and SA, Chandra S, McGarrell DM, Schmidt TM, Garrity Sarah Flood Page for assistance with sequence editing and GM, Tiedje JM (2003) The RibosomalDatabase analysis. We also thank K. Kalbitz and two anonymous Project (RDP-II): previewing a new autoaligner reviewers for valuable comments on the manuscript. This that allows regularupdates and the new prokaryotic work was supported by National Science Foundation taxonomy. Nucleic Acids Res 31:442-443 (NSF) Grant DEB-0089447 (to A.R.T. and S.K.S.) and Davidson EA, Janssens lA (2006) Temperature sensitivity by NSF Grant DEB-0515744 (to A.R.T. and C.C.C.) of soil carbon decomposition and feedbacks to climate change. Nature 440:165-173 Don A, Kalbitz K (2005) Amounts and degradability References of dissolved organic carbon from foliar litter at different decomposition stages. Soil Biol Biochem Abd-EI-Haleem D, Moawad H, Zaki EA, Zaki S (2002) 37:2171-2179 Molecular characterization of phenol-degrading bac­ Fang C, Smith P, Smith JU, Moncrief JB (2005) Incorpo­ teria isolated from different Egyptian ecosystems. rating microorganisms as decomposers into models to Microb Ecol 43:217-224 simulate soil organic matter decomposition. Geoder­ Adams GA, Wall DH (2000) Biodiversity above and ma 129:139-146 below the surface of soils and sediments: linkages Fierer N, Bradford MA, Jackson RB (2007) Towards an and implications for global change. Bioscience 50: ecological classification of soil bacteria. Ecology (in 1043-1049 press)

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Grayston SJ, Wang S, Campbell CD, Edwards AC (1998) sensitivity to litter chemistry, texture and management. Selective influence of plant species on microbial In: Bryant RB, Arnold RW (eds) Quantitative diversity in the rhizosphere. Soil Biol Biochem modeling of soil forming processes. Soil Science 30:369-378 Society of America Special Publication 39, Madison, Holdridge LR, Grenke WC, Hatheway WH, Liang T, Tosi pp 147-167 JA (eds) (1971) Forest Environments in Tropical Life Potts DL, Huxman TE, Cable JM, English NB, Ignace Zones: a pilot study. Pergamon, Oxford DD, Eilts JA, Mason Ml, Weltzin IF, Williams DC Hooper DU, Bignell DE, Brown VK, Brussaard L, (2006) Antecedent moisture and seasonal precipita­ Dangerfleld JM, Wall DH, Wardle DL, Coleman tion influence the response of canopy-scale carbon DC, Ciller KE, Lavelle P, Van der Putten WH, De and water exchange to rainfall pulses in a semi-arid Ruiter PC, Rusek J, Silver WL, Tiedje JM, Wolters V grassland. New Phytol 170:849-860 (2000) Interactions between aboveground and below- Raich JW, Schlesinger WH (1992) The global carbon ground biodiversity in terrestrial ecosystems: Patterns, dioxide flux in soil respiration and its relationships to mechansims, and feedbacks. Bioscience 50:1049-1061 vegetation and climate. Tellus 44B:81-99 Housman DC, Naumburg E, Huxman TE, Charlet TN, Rodriguez H, Fraga R (1999) Phosphate solubilizing Nowak RS, Smith SC (2006) Increases in desert shrub bacteria and their role in plant growth promotion. productivity under elevated carbon dioxide vary with Biotechnol Adv 17:319-339 water availability. Ecosystems 9:374-385 Sacks WJ, Schimel DS, Monson RK, Braswell BH (2006)

Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a Model-data synthesis of diurnal and seasonal 2 CQ program to detect chimeric sequences in multiple fluxes at Niwot Ridge, Colorado. Global Change Biol sequence alignments. Bioinformatics 20:2317-2319 12:240-259 Jackson CR, Harper IP, Willoughby D, Roden BE, Sambrook J, Russell DW (2001) Molecular cloning: a Churchill PF (1997) A simple, efficient method for laboratory manual. Cold Spring Harbor Laboratory the separation of humic substances and DNA from Press, Cold Spring Harbor environmental samples. AppI Environ Microbiol Schlesinger WH, Andrews JA (2000) Soil respiration and 63:4993-4995 the global carbon cycle. Biogeochemistry 48:7-20 Lane DJ (1991) 16S/23S rRNA Sequencing. In: Stacke- Schloss PD, Handelsman J (2005) Introducing DQTUR, a brandt E, Goodfellow M (eds) Nucleic acid tech­ computer program for defining operational taxonomic niques in bacterial systematics. Wiley, Chichester, units and estimating species richness. AppI Environ pp 115-175 Microbiol 71:1501-1506 Lloyd J, Taylor JA (1994) On the temperature dependence Schmidt SK, Costello EK, Nemergut DR, Cleveland CC, of soil respiration. Funct Ecol 8:315-323 Reed SC, Weintraub MN, Meyer AF, Martin AM Ludwig W, Strunk O, Westram R et al (2004) ARB: a (2007) Microbial turnover and seasonal succession software environment for sequence data. Nucleic drive biogeochemical cycles in the alpine. Ecology (in Acids Res 32:1363-1371 press) Meentemeyer V (1978) Macroclimate and lignin control of Schwendenmann L, Veldkamp E, Brenes T, Q’Brien JJ, litter decomposition rates. Ecology 59:465^72 Mackensen J (2003) Spatial and temporal variation in

Monson RK, Lipson DL, Burns SP, Turnipseed AA, Delany soil CQ 2 efflux in an old-growth neotropical rain AC, Williams MW, Schmidt SK (2006) Winter forest forest. La Selva, Costa Rica. Biogeochemistry 64:111- soil respiration controlled by climate and microbial 128 community composition. Nature 439:711-714 Schwendenmann L, Veldkamp E (2005) The role of More MI, Herrick JB, Silva MC, Ghiorse WC, Madsen EL dissolved organic carbon, dissolved organic nitrogen, (1994) Quantitative cell-Iysis of indigenous microor­ and dissolved inorganic nitrogen in a tropical wet ganisms and rapid extraction of microbial DNA from forest ecosystem. Ecosystems 8:339-351 sediment. AppI Environ Microbiol 60:1572-1580 Scott-Denton LE, Sparks KL, Monson RK (2003) Spatial Neff JC, Asner CP (2001) Dissolved organic carbon in and temporal controls of soil respiration rate in a terrestrial ecosystems: synthesis and a model. Ecosys­ high-elevation, subalpine forest. Soil Biol Biochem tems 4:29-48 35:525-534 Niisslein K, Tiedje JM (1999) Soil bacterial community Scott-Denton LE, Rosenstiel TN, Monson RK (2006) shift correlated with change from forest to pasture Differential controls by climate and substrate over the vegetation in a tropical soil. AppI Environ Microbiol heterotrophic and rhizospheric components of soil 65:3622-3626 respiration. Global Change Biol 12:205-216 Padmanabhan P, Padmanabhan S, DeRito C et al (2003) Snedecor GW, Cochran WC (1989) Statistical Methods. Respiration of ^^C-Iabeled substrates added to soil in Iowa State University, Ames the field and subsequent 16S rRNA gene analysis of Souchie EL, Saggin-Junior QJ, Silva EMR et al (2006) ^^C-Iabeled soil DNA. AppI Environ Microbiol Communities of P-soIubilizing bacteria, fungi and 69:1614-1622 arbuscular mycorrhizal fungi in grass pasture and Parton WJ, Schimel DS, Cole CV, Ojima DS (1994) A secondary forest of Paraty, RJ-Brazil. An Acad Braz general model for soil organic matter dynamics: Cienc 78:183-193

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