- Email: [email protected]
Microbial diversity limits soil heterotrophic respiration and mitigates the respiration response to moisture increase
Soil Biology & Biochemistry 98 (2016) 180e185
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Microbial diversity limits soil heterotrophic respiration and mitigates the respiration response to moisture increase Fen-Guo Zhang a, Quan-Guo Zhang b, * a
College of Life Sciences, Shanxi Normal University, Linfen, Shanxi 041004, China State Key Laboratory of Earth Surface Processes and Resource Ecology and MOE Key Laboratory for Biodiversity Science and Ecological Engineering, Beijing Normal University, Beijing 100875, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 December 2015 Received in revised form 20 April 2016 Accepted 24 April 2016 Available online 29 April 2016
Decline in soil biodiversity associated with anthropogenic activities has raised concerns about the consequences for ecosystem functions. It remains uncertain how important soil microbial diversity is relative to abiotic factors, and how they interact, in driving ecosystem processes. Here we present results of a soil microcosm experiment in which microbial diversity and moisture conditions were independently manipulated. Loss of microbial diversity led to higher rates of soil microbial respiration, and the diversity effect was maintained over time during the course of the experiment. Higher moisture also enhanced soil respiration; but the moisture effect reduced over time, more rapidly in microcosms of higher microbial diversity. Overall, loss of microbial diversity enhanced soil respiration to a greater extent than moisture elevation, and also exacerbated the response of soil respiration to water addition. Loss of negative species interactions in microcosms of lower microbial diversity might be the major reason for the diversity effects observed in this study. Our results suggest that the integrity of soil microbial communities be crucial for the maintenance of soil carbon storage function. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Biodiversity Ecosystem functioning Global change Microbiome Soil respiration
1. Introduction Loss of species diversity may cause significant changes in ecosystem functions, with its effects being of comparable magnitude of that of many abiotic environmental factors including nutrient availability and temperature (Tilman, 1999; Loreau et al., 2001; Reich et al., 2001; Hooper et al., 2012; Tilman et al., 2012; Allison et al., 2013; Eisenhauer et al., 2013; Boyero et al., 2014; Bradford et al., 2014; Steinauer et al., 2014). Meanwhile, species diversity may interact with the abiotic factors as drivers of ecosystem processes. Species diversity may, on one hand, alleviate the impacts of stressful environmental factors such as drought (Tilman and Downing, 1994; Yachi and Loreau, 1999; Mulder et al., 2001; Gonzalez and Loreau, 2009; Awasthi et al., 2014), and on the other hand, enhance ecosystem responses to ‘positive’ perturbations including nutrient enrichment (Reich et al., 2001; He et al., 2002; Fridley, 2003). This can result from a ‘sampling effect’ as more diverse communities are more likely to contain particular
* Corresponding author. E-mail addresses: [email protected] (F.-G. Zhang), [email protected] (Q.-G. Zhang). http://dx.doi.org/10.1016/j.soilbio.2016.04.017 0038-0717/© 2016 Elsevier Ltd. All rights reserved.
species that are very resistant to environmental stress or highly responsive to improved conditions (Aarssen, 1997; Huston, 1997), or niche complementarity and facilitation among species because wider ranges of functional traits in species-richer communities can positively affect ecosystem performance under novel environmental conditions (Tilman et al., 1997; Loreau, 2000). Microbes, particularly those inhabiting topsoil, are characterized by tremendous diversity which is now threatened by anthropogenic activities including agricultural intensification and land use changes (Maeder et al., 2002; de Vries et al., 2013; Paula et al., 2014). While it seems reasonable to expect functional redundancy among hyper-diverse microbes (Torsvik et al., 2002; Nannipieri et al., 2003; Allison and Martiny, 2008; Cleveland et al., 2014), experimental studies that manipulated microbial communities often, although not always, found negative consequences of microbial diversity loss for ecological functions such as nitrogen cycling or biomass production (Degens, 1998; Wertz et al., 2007; Hol et al., 2010; Peter et al., 2011; van Elsas et al., 2012; Philippot et al., 2013; Wagg et al., 2014). However, it remains uncertain how important microbial diversity is relative to abiotic factors, and how they interact, in driving ecosystem processes (but see Degens, 1998; Wertz et al., 2007; Awasthi et al., 2014).
F.-G. Zhang, Q.-G. Zhang / Soil Biology & Biochemistry 98 (2016) 180e185
Here we experimentally address the dependency of soil microbial respiration on microbial diversity and soil moisture. Knowledge of the determinants of soil microbial respiration is crucial for a better understanding of global carbon cycles under future climatic conditions (Spehn et al., 2000; Curiel Yuste et al., 2007; Curiel Yuste et al., 2010; Suseela et al., 2012; Zhou et al., 2012; Li et al., 2013). There has been a rich literature on how abiotic factors, including temperature and moisture, affect soil respiration; however, microbial diversity and composition have often been confounding factors as they co-vary with the abiotic factors (Carney et al., 2007; Cleveland et al., 2007; Allison et al., 2013; Karhu et al., 2014; Matulich and Martiny, 2014; Whitaker et al., 2014; GarcíaPalacios et al., 2015). It is possible that higher species diversity allows microbial communities to achieve more reliable ecosystem functions under different moisture regimes. We carried out a microcosm experiment where microbial diversity and soil moisture were manipulated independently, to address their interaction in driving soil heterotrophic respiration.
2. Materials and methods 2.1. Soil collection and treatment Soil was collected from the top 15 cm of a semiarid grassland (Xilingol, Inner Mongolia, China; 43 320 N, 116 300 E), where Leymus chinensis was the dominant plant species. The soil was sieved to <2 mm, homogenized, and divided into microcosms of 160 g (equivalent dry mass) in 250 mL Schott Duran bottles. These microcosms were sterilized by 100 kGy gamma irradiation (Hongyisifang radiation technology Co., Ltd, Beijing, China), with soil sterility checked by enumeration of culturable bacteria on nutrient agar plates (3 g L1 beef extract, 10 g L1 peptone, 5 g L1 NaCl and 15 g L1 agar). Microcosms were then inoculated with suspensions of the same soil that had not been sterilized. Inocula were prepared by homogenizing 50 g of soil (equivalent dry mass) in 100 mL of sterile demineralized water by grinding and vortexing, followed by serial dilutions in sterile demineralized water. Sterile soil microcosms were inoculated with dilutions to create inocula equivalent to 102, 104, and 106 g of non-sterile soil per g sterile soil, 18 replicates per treatment level. Soil microcosms were incubated for recovery for 1 month at 25 C (which is near the mean topsoil temperature at the soil collection site in June and July), with bottle lids loosened and moisture content adjusted to 10% gravimetric water content (water/dry soil; near the moisture content of our source soil) regularly by addition of sterile water. Previous studies showed that the dilution disturbance could lead to a significant decrease in microbial diversity (Wertz et al., 2006; Peter et al., 2011; van Elsas et al., 2012). In the present study, experimental microcosms were incubated for recovery for one month, a period that is likely long enough for a full recovery in microbial activity (Zhang and Zhang, 2015). Analyses of soil property and microbial composition were carried out immediately after the 1-month recovery incubation (with six replicate microcosms randomly chosen from each level of dilution treatment for the measurements). Microcosms recovering from different levels of dilution treatments showed no detectable difference in total carbon or nitrogen content (Appendix A: Methods A1, Fig. A1). Pyrosequencing analyses of bacterial and fungal species composition (see detailed experimental procedures in Appendix A: Methods A2) confirmed that increasing levels of dilution treatments led to progressively decreased bacterial and fungal diversity (Fig. 1; Figs. A2 and A3). The microcosms under the 102, 104, and 106 dilution treatments are hereafter referred to as high-, intermediate- and low-diversity microcosms, respectively.
181
2.2. Experimental setup and measurement of soil respiration After the 1-month recovery growth, microcosms were incubated for another 46 days under three different moisture conditions. The 18 microcosms at each level of microbial diversity were randomly assigned into three groups, six replicates per group. Each group was exposed to one of the following three moisture treatments: low (10% gravimetric water content, near 30% of the waterholding capacity), high (20%), or fluctuating moisture (changing between 10% and 20%). For the low- or high-moisture microcosms, water content was maintained at 10% or 20% by adding sterile water at a 4-day interval (at day 1, 5, etc.). For the fluctuating-moisture microcosms, moisture content was increased to 20% at day 1. Then moisture was measured at a 4-day interval; when the average moisture of the microcosms became <10%, moisture of every microcosm was increased abruptly to 20% (pulse water addition was performed at day 1, 17 and 37; Fig. A4). Respiration rate of each microcosm was measured at a 4-day interval (at day 2, 6, etc.), with a Li-Cor 840A CO2/H2O analyzer (Li-Cor Environmental, Lincoln, NE, USA). Before measurement, each microcosm (bottle) was placed in water bath at 25 C and sealed with a rubber stopper. The rubber stopper had two ports that were connected with the gas analyzer, thus the bottle and the analyzer became a closed system. The analyzer maintained an air flow through this closed system, and recorded temperature, air pressure, CO2 and water content every second. For each microcosm, we ran the measurement for 70 s; data from 31 to 70 s were used for CO2 efflux calculation (fluctuations in air pressure and CO2 concentration may be observed at the beginning of the 70-s measurement, but then air pressure would remain constant and CO2 concentration increases gradually and linearly). The slope of CO2 concentration against time (dCO2/dt) was calculated, and the amount of CO2 evolved from the soil per second is the product of the slope and the volume of the system. The volume of the system was measured by injecting a known quantity of air with known CO2 concentration into the system that had been CO2-free (by flushing N2) and measuring the final CO2 concentration, with the dilution factor in CO2 concentration equal to the ratio of injected air volume to the system volume (Curiel Yuste et al., 2007). Soil respiration rate was expressed as mmol per g dry soil per second.
2.3. Data analysis There were three explanatory variables for soil respiration rates: level of microbial diversity, moisture treatment, and time. However, time may not be a meaningful explanatory variable for the fluctuating-moisture microcosms, as its effect can be confounded with that of moisture changes. To avoid this issue, we carried out two separate analyses. First, the average respiration rate over time was calculated for each microcosm, and analyzed using ANOVA, with diversity level and moisture treatment as two categorical explanatory variables. Second, respiration rates of the low- and high-moisture microcosms at all points in time were analyzed using linear mixed-effects model, with diversity level and moisture treatment as two categorical explanatory variables, time as a continuous explanatory variable, and microcosm ID as a random factor.
2.4. Accession numbers The pyrosequencing data have been deposited in NCBI Sequence Read Archive under accession number SRP057044.
182
F.-G. Zhang, Q.-G. Zhang / Soil Biology & Biochemistry 98 (2016) 180e185
Fig. 1. Species (OTUs) richness, Pielou's evenness and Shannon index of bacterial and fungal communities in a subset of experimental microcosms, after the 1-month recovery growth. Data show mean ± SE (n ¼ 6). Data points with the same letter within each panel do not significantly differ (Tukey multiple comparisons, Padj > 0.05). Fungal diversity indices in microcosms under 106 dilution treatment have been excluded from analysis, as in four out of the six microcosms PCR amplification for fungal communities was unsuccessful (note that a failure in amplification does not mean that the fungal abundance or diversity was zero).
3. Results 3.1. The effect of dilution treatments on soil microbial diversity A progressive decrease in microbial diversity with increasing magnitude of dilution treatment was observed, inferred by pyrosequencing analyses of bacterial and fungal communities in microcosms immediately after the 1-month recovery incubation. Compared with the microcosms of 102 dilution treatment, those under 104 treatment had a 35% decrease in bacterial species richness and a 31% decrease in fungal species richness, and those under 106 treatment had a 70% decrease in bacterial species richness (assays of fungal diversity failed; Fig. 1). 3.2. Overall effects of microbial diversity and moisture Analysis of the respiration rates averaged over time suggested a positive effect of microbial diversity loss on respiration (F2,45 ¼ 74.5, P < 0.001; Fig. 2d). Respiration rates of the intermediate- and lowdiversity microcosms were 48% and 61% higher than that of the
high-diversity microcosms, respectively. Soil respiration also showed a significant response to moisture content (F2,45 ¼ 15.3, P < 0.001), with high- and fluctuating-moisture treatments increasing respiration rates by 14% and 24%, respectively. There was a significant diversity moisture interaction effect (F4,45 ¼ 6.08, P < 0.001); high- and fluctuating-moisture treatments led to an increase in the rate of respiration in the intermediate- and lowdiversity microcosms, but not in the high-diversity microcosms (Fig. 2d). 3.3. Temporal changes in diversity and moisture effects Analysis of all repeated measures of respiration rates for the low- and high-moisture microcosms indicated significant effects of microbial diversity (F2,30 ¼ 54.2, P < 0.001), moisture treatment (low versus high moisture, F1,30 ¼ 15.6, P < 0.001) and diversity moisture interaction (F2,30 ¼ 13.6, P < 0.001; Fig. 2). Respiration rates did not show an overall trend over time (F1,389 ¼ 1.84, P ¼ 0.176). The effect of diversity time interaction was not significant (F2,389 ¼ 2.45, P ¼ 0.088), suggesting a persistent
F.-G. Zhang, Q.-G. Zhang / Soil Biology & Biochemistry 98 (2016) 180e185
183
lower microbial diversity (Fig. 2). 4. Discussion
Fig. 2. Respiration rates of soil microcosms under different diversity and moisture treatments. Both repeated measures throughout the experiment (aec) and averages over time (d) are shown. Within each diversity level in panel (d), bars with the same letter do not significantly differ (Tukey multiple comparisons, Padj > 0.05). Data show mean ± SE (n ¼ 6).
diversity effect on soil respiration over time. There was a significant moisture time interaction effect (F1,389 ¼ 78.8, P < 0.001), and a significant diversity moisture time interaction effect (F2,389 ¼ 3.72, P ¼ 0.025). Specifically, the high-moisture treatment enhanced respiration at the beginning of the experiment, regardless of microbial diversity level; then the moisture effect reduced over time, and the fade of this effect occurred very rapidly in the high-diversity microcosms and more slowly in microcosms of
Here we manipulated soil microbial diversity using a dilutionto-extinction approach. Microcosms recovering from different magnitudes of dilution treatments attained different levels of microbial diversity. We expected that a relatively short period (one month) of recovery incubation, on one hand, could allow a full recovery in microbial activity, and on the other hand, would not cause substantial difference in soil properties among microcosms of different levels of microbial diversity. The two assumptions have very likely held true. The fact that respiration rates of our microcosms during the main experiment did not show an overall trend over time suggests that the microbial activity had reached a stationary state during the recovery incubation; and the maintenance of diversity effect on soil respiration over time during the course of the main experiment implies that soil organic carbon content had not been decreased to a very low level during the recovery incubation to be the limiting factor for soil CO2 flux. Furthermore, we also found that our microcosms following the 1-month recovery incubation did not show significant difference in total carbon or nitrogen content among different microbial diversity treatments (Fig. A1). In our experiment, high microbial diversity and low moisture content represented more natural conditions. Both microbial diversity decline and moisture elevation caused higher rates of soil microbial respiration. The magnitude of the diversity effect on soil respiration was very large relative to the moisture effect. For instance, the intermediate-diversity microcosms, with a <40% decrease in bacterial or fungal species richness relative to the highdiversity microcosms (Fig. 1), had a ~48% increase in respiration rates (Fig. 2); the high-moisture treatments (with a 100% increase in moisture compared with the low-moisture treatment) increased respiration rates by only ~14% (Fig. 2). This is consistent with a previous suggestion that species diversity can show stronger effects on ecosystem processes than certain abiotic factors (Eisenhauer et al., 2013; Steinauer et al., 2014). Our finding that soil biodiversity limits soil respiration is consistent with a previous study (Bradford et al., 2014). We also found that loss of microbial diversity could exacerbate the responsiveness of soil respiration to moisture elevation. The high-diversity microcosms observed a rapid reduction over time in the enhancement effect of soil respiration by moisture elevation; in microcosms of lower microbial diversity, however, the moisture effect on soil respiration persisted longer during the experiment (Fig. 2). There are two possible explanations for the negative effect of microbial diversity on soil respiration. First, negative species interactions may limit decomposers' activity, and these species interactions may have been impaired in lower-diversity microcosms. One example is interference competition, as certain microbes may produce toxins that depress the growth of other species, or produce proteins that slow organic matter degradation by, e.g., contributing to micro-aggregate formation in soils (King, 2011). Trophic interactions may also negatively impact decomposers; for instance, top-down control of soil bacteria by viruses can slow soil heterotrophic respiration (Allen et al., 2010). Second, species-rich communities may contain particular species that had poor contributions to ecosystem functions but can somehow attain dominance. This is called ‘negative sampling effect’, which has been suggested to be common for non-biomass functions (Jiang et al., 2008). In the present study, microcosms exposed to higher dilution treatments may have shown a reduction in species diversity as well as a shift in species composition toward dominance by fastgrowing species. This is because the dilution treatment not only
184
F.-G. Zhang, Q.-G. Zhang / Soil Biology & Biochemistry 98 (2016) 180e185
removed species of very low abundances, but also decreased community sizes, thus species capable of rapid growth may have enjoyed a population growth advantage during the recovery incubation. The fast-growing species are likely to have higher respiration rates but lower carbon use efficiency. In particular, the abundances of fungal species involved in slower decomposition pathways (Wardle et al., 2004; Fierer et al., 2009; Strickland et al., 2009; de Graaff et al., 2010) may have decreased relative to bacteria in microcosms of lower microbial diversity (suggested by the fact that PCR assays of fungal communities have failed for low-diversity microcosms). Within bacteria, an increase in the abundances of Proteobacteria, and a decrease in Actinobacteria, were observed in lower-diversity microcosms (Fig. A2), the former being active decomposers, and the latter showing slow-growing behavior (Padmanabhan et al., 2003; Fierer et al., 2007). Either a negative sampling effect or negative species interactions may explain an overall negative diversity effect on soil respiration. For the interaction effect of microbial diversity and moisture, however, the negative species interactions hypothesis provide more likely explanations. Enhancement of decomposers' activity by improved moisture conditions can be depressed by a follow-up increase in the negative species interactions. For instance, moisture increase can immediately cause more active bacterial or fungal growth and thus increase the rate of soil respiration, but would also lead to further accumulation of proteins that limit decomposition, or an increase in the abundances of predators of the decomposers (Tuomi et al., 1995). The intermediate- and low-diversity microcosms may have lost a majority of the negative species interactions, allowing the enhancement effect by moisture elevation to persist longer. An explanation based on negative sampling effect appears less likely, which would be conditioned on (i) that functionally poor decomposers dominated high-diversity communities and (ii) that the poor decomposers also showed more rapid physiological acclimation to higher-moisture conditions. The fluctuating-moisture microcosms had insignificantly higher soil respiration rates relative to the high-moisture microcosms (Fig. 2) although the former had overall lower water content. Every episode of abrupt moisture elevation could stimulate soil respiration, maintaining a ‘Birch effect’, i.e., transient increase in CO2 flux following soil rewetting (Birch, 1964; Unger et al., 2010). This implies that the reduction of respiration rate over time in the highmoisture microcosms mainly results from biotic factors including negative species interaction, rather than resource or water limitation. It is possible that, in the fluctuating-moisture microcosms, factors slowing soil respiration during the high-moisture periods lost their effects when moisture became lower and the later pulse of moisture elevation could stimulate respiration once again. Our findings appear to contradict several recent studies that showed positive diversity effects on microbial processes (Wertz et al., 2007; Peter et al., 2011; van Elsas et al., 2012; de Vries et al., 2013; Philippot et al., 2013; Wagg et al., 2014). One possible explanation is that we measured a non-biomass function (CO2 flux) directly but some earlier studies actually measured biomass production functions or used biomass-based measurements to infer ecosystem processes (e.g. using copy number of certain genes to estimate the rate of nitrate oxidation), while biomass and nonbiomass functions may show different forms of dependence on species diversity (Jiang et al., 2008). The present study specifically suggests that loss of microbial diversity can increase the rate of soil microbial respiration and exacerbate the responsiveness of soil respiration to improved moisture conditions. It is of interest in future to investigate whether microbial diversity mediate the response of soil ecosystem functions to other types of environmental changes, in particular, labile carbon input that may lead to
priming effect on soil organic matter mineralization (Reinsch et al., 2013; Rousk et al., 2015). Our results imply that loss of microbial diversity may impair the soil carbon storage function under environmental changes that is currently a major concern (Carney et al., 2007; Cleveland et al., 2007; Zhou et al., 2007; Curiel Yuste et al., 2010; Suseela et al., 2012; Karhu et al., 2014; Wang et al., 2014). Therefore, among the management strategies for terrestrial carbon storage, those effectively protecting the integrity of soil microbial communities might be the most promising. Acknowledgements We thank O. Petchey for his helpful comments on the manuscript. This study was funded by the National Natural Science Foundation of China (31222010 and 31421063) and the 111 project (B13008). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2016.04.017. References Aarssen, L.W., 1997. High productivity in grassland ecosystems: effected by species diversity or productive species? Oikos 80, 183e184. Allen, B., Willner, D., Oechel, W.C., Lipson, D., 2010. Top-down control of microbial activity and biomass in an Arctic soil ecosystem. Environ. Microbiol. 12, 642e648. Allison, S.D., Lu, Y., Weihe, C., Goulden, M.L., Martiny, A.C., Treseder, K.K., Martiny, J.B.H., 2013. Microbial abundance and composition influence litter decomposition response to environmental change. Ecology 94, 714e725. Allison, S.D., Martiny, J.B.H., 2008. Resistance, resilience, and redundancy in microbial communities. Proc. Natl. Acad. Sci. U. S. A. 105, 11512e11519. Awasthi, A., Singh, M., Soni, S.K., Singh, R., Kalra, A., 2014. Biodiversity acts as insurance of productivity of bacterial communities under abiotic perturbations. ISME J. 8, 2445e2452. Birch, H.F., 1964. Mineralisation of plant nitrogen following alternate wet and dry conditions. Plant Soil 20, 43e49. Boyero, L., Cardinale, B.J., Bastian, M., Pearson, R.G., 2014. Biotic vs. abiotic control of decomposition: a comparison of the effects of simulated extinctions and changes in temperature. PLoS ONE 9, e87426. Bradford, M.A., Wood, S.A., Bardgett, R.D., Black, H.I.J., Bonkowski, M., Eggers, T., Grayston, S.J., Kandeler, E., Manning, P., Set€ al€ a, H., Jones, T.H., 2014. Discontinuity in the responses of ecosystem processes and multifunctionality to altered soil community composition. Proc. Natl. Acad. Sci. U. S. A. 111, 14478e14483. Carney, K.M., Hungate, B.A., Drake, B.G., Megonigal, J.P., 2007. Altered soil microbial community at elevated CO2 leads to loss of soil carbon. Proc. Natl. Acad. Sci. U. S. A. 104, 4990e4995. Cleveland, C.C., Nemergut, D.R., Schmidt, S.K., Townsend, A.R., 2007. Increases in soil respiration following labile carbon additions linked to rapid shifts in soil microbial community composition. Biogeochemistry 82, 229e240. Cleveland, C.C., Reed, S., Keller, A., Nemergut, D., O'Neill, S., Ostertag, R., Vitousek, P., 2014. Litter quality versus soil microbial community controls over decomposition: a quantitative analysis. Oecologia 174, 283e294. Curiel Yuste, J., Baldocchi, D.D., Gershenson, A., Goldstein, A., Misson, L., Wong, S., 2007. Microbial soil respiration and its dependency on carbon inputs, soil temperature and moisture. Glob. Change Biol. 13, 2018e2035. Curiel Yuste, J., Ma, S., Baldocchi, D.D., 2010. Plant-soil interactions and acclimation to temperature of microbial-mediated soil respiration may affect predictions of soil CO2 efflux. Biogeochemistry 98, 127e138. de Graaff, M.-A., Classen, A.T., Castro, H.F., Schadt, C.W., 2010. Labile soil carbon inputs mediate the soil microbial community composition and plant residue decomposition rates. New Phytol. 188, 1055e1064. bault, E., Liiri, M., Birkhofer, K., Tsiafouli, M.A., Bjørnlund, L., Bracht de Vries, F.T., The Jørgensen, H., Brady, M.V., Christensen, S., de Ruiter, P.C., d'Hertefeldt, T., Frouz, J., Hedlund, K., Hemerik, L., Hol, W.H.G., Hotes, S., Mortimer, S.R., Set€ al€ a, H., Sgardelis, S.P., Uteseny, K., van der Putten, W.H., Wolters, V., Bardgett, R.D., 2013. Soil food web properties explain ecosystem services across European land use systems. Proc. Natl. Acad. Sci. U. S. A. 110, 14296e14301. Degens, B.P., 1998. Decreases in microbial functional diversity do not result in corresponding changes in decomposition under different moisture conditions. Soil Biol. Biochem. 30, 1989e2000. Eisenhauer, N., Dobies, T., Cesarz, S., Hobbie, S.E., Meyer, R.J., Worm, K., Reich, P.B., 2013. Plant diversity effects on soil food webs are stronger than those of elevated CO2 and N deposition in a long-term grassland experiment. Proc. Natl. Acad. Sci. U. S. A. 110, 6889e6894.
F.-G. Zhang, Q.-G. Zhang / Soil Biology & Biochemistry 98 (2016) 180e185 Fierer, N., Bradford, M.A., Jackson, R.B., 2007. Toward an ecological classification of soil bacteira. Ecology 88, 1354e1364. Fierer, N., Strickland, M.S., Liptzin, D., Bradford, M.A., Cleveland, C.C., 2009. Global patterns in belowground communities. Ecol. Lett. 12, 1238e1249. Fridley, J.D., 2003. Diversity effects on production in different light and fertility environments: an experiment with communities of annual plants. J. Ecol. 91, 396e406. García-Palacios, P., Vandegehuchte, M.L., Shaw, E.A., Dam, M., Post, K.H., Ramirez, K.S., Sylvain, Z.A., de Tomasel, C.M., Wall, D.H., 2015. Are there links between responses of soil microbes and ecosystem functioning to elevated CO2, N deposition and warming? A global perspective. Glob. Change Biol. 21, 1590e1600. Gonzalez, A., Loreau, M., 2009. The causes and consequences of compensatory dynamics in ecological communities. Annu. Rev. Ecol. Evol. Syst. 40, 393e414. He, J.S., Bazzaz, F.A., Schmid, B., 2002. Interactive effects of diversity, nutrients and elevated CO2 on experimental plant communities. Oikos 97, 337e348. Hol, W.H.G., de Boer, W., Termorshuizen, A.J., Meyer, K.M., Schneider, J.H.M., van Dam, N.M., van Veen, J.A., van der Putten, W.H., 2010. Reduction of rare soil microbes modifies plant-herbivore interactions. Ecol. Lett. 13, 292e301. Hooper, D.U., Adair, E.C., Cardinale, B.J., Byrnes, J.E.K., Hungate, B.A., Matulich, K.L., Gonzalez, A., Duffy, J.E., Gamfeldt, L., O'Connor, M.I., 2012. A global synthesis reveals biodiversity loss as a major driver of ecosystem change. Nature 486, 105e108. Huston, M.A., 1997. Hidden treatments in ecological experiments: re-evaluating the ecosystem function of biodiversity. Oecologia 110, 449e460. Jiang, L., Pu, Z., Nemergut, D.R., 2008. On the importance of the negative selection effect for the relationship between biodiversity and ecosystem functioning. Oikos 117, 488e493. Karhu, K., Auffret, M.D., Dungait, J.A.J., Hopkins, D.W., Prosser, J.I., Singh, B.K., Subke, J.-A., Wookey, P.A., Agren, G.I., Sebastia, M.-T., Gouriveau, F., Bergkvist, G., Meir, P., Nottingham, A.T., Salinas, N., Hartley, I.P., 2014. Temperature sensitivity of soil respiration rates enhanced by microbial community response. Nature 513, 81e84. King, G.M., 2011. Enhancing soil carbon storage for carbon remediation: potential contributions and constraints by microbes. Trends Microbiol. 19, 75e84. Li, D., Zhou, X., Wu, L., Zhou, J., Luo, Y., 2013. Contrasting responses of heterotrophic and autotrophic respiration to experimental warming in a winter annualdominated prairie. Glob. Change Biol. 19, 3553e3564. Loreau, M., 2000. Biodiversity and ecosystem functioning: recent theoretical advances. Oikos 91, 3e17. Loreau, M., Naeem, S., Inchausti, P., Bengtsson, J., Grime, J.P., Hector, A., Hooper, D.U., Huston, M.A., Raffaelli, D., Schmid, B., Tilman, D., Wardle, D.A., 2001. Biodiversity and ecosystem functioning: current knowledge and future challenges. Science 294, 804e808. Maeder, P., Fliessbach, A., Dubois, D., Gunst, L., Fried, P., Niggli, U., 2002. Soil fertility and biodiversity in organic farming. Science 296, 1694e1697. Matulich, K.L., Martiny, J.B.H., 2014. Microbial composition alters the response of litter decomposition to environmental change. Ecology 96, 154e163. Mulder, C.P.H., Uliassi, D.D., Doak, D.F., 2001. Physical stress and diversityproductivity relationships: the role of positive interactions. Proc. Natl. Acad. Sci. U. S. A. 98, 6704e6708. Nannipieri, P., Ascher, J., Ceccherini, M.T., Landi, L., Pietramellara, G., Renella, G., 2003. Microbial diversity and soil functions. Eur. J. Soil Sci. 54, 655e670. Padmanabhan, P., Padmanabhan, S., DeRito, C., Gray, A., Gannon, D., Snape, J.R., Tsai, C.S., Park, W., Jeon, C., Madsen, E.L., 2003. Respiration of 13C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of C-13labeled soil DNA. Appl. Environ. Microbiol. 69, 1614e1622. Paula, F.S., Rodrigues, J.L.M., Zhou, J., Wu, L., Mueller, R.C., Mirza, B.S., Bohannan, B.J.M., Nüsslein, K., Deng, Y., Tiedje, J.M., Pellizari, V.H., 2014. Land use change alters functional gene diversity, composition and abundance in Amazon forest soil microbial communities. Mol. Ecol. 23, 2988e2999. Peter, H., Beier, S., Bertilsson, S., Lindstrom, E.S., Langenheder, S., Tranvik, L.J., 2011. Function-specific response to depletion of microbial diversity. ISME J. 5, 351e361. Philippot, L., Spor, A., Henault, C., Bru, D., Bizouard, F., Jones, C.M., Sarr, A., Maron, P.A., 2013. Loss in microbial diversity affects nitrogen cycling in soil. ISME J. 7, 1609e1619. Reich, P.B., Knops, J., Tilman, D., Craine, J., Ellsworth, D., Tjoelker, M., Lee, T., Wedin, D., Naeem, S., Bahauddin, D., Hendrey, G., Jose, S., Wrage, K., Goth, J., Bengston, W., 2001. Plant diversity enhances ecosystem responses to elevated CO2 and nitrogen deposition. Nature 410, 809e812. Reinsch, S., Ambus, P., Thornton, B., Paterson, E., 2013. Impact of future climatic
185
conditions on the potential for soil organic matter priming. Soil Biol. Biochem. 65, 133e140. Rousk, J., Hill, P.W., Jones, D.L., 2015. Priming of the decomposition of ageing soil organic matter: concentration dependence and microbial control. Funct. Ecol. 29, 285e296. Spehn, E.M., Joshi, J., Schmid, B., Alphei, J., Korner, C., 2000. Plant diversity effects on soil heterotrophic activity in experimental grassland ecosystems. Plant Soil 224, 217e230. Steinauer, K., Tilman, D., Wragg, P.D., Cesarz, S., Cowles, J.M., Pritsch, K., Reich, P.B., Weisser, W.W., Eisenhauer, N., 2014. Plant diversity effects on soil microbial functions and enzymes are stronger than warming in a grassland experiment. Ecology 96, 99e112. Strickland, M.S., Osburn, E., Lauber, C., Fierer, N., Bradford, M.A., 2009. Litter quality is in the eye of the beholder: initial decomposition rates as a function of inoculum characteristics. Funct. Ecol. 23, 627e636. Suseela, V., Conant, R.T., Wallenstein, M.D., Dukes, J.S., 2012. Effects of soil moisture on the temperature sensitivity of heterotrophic respiration vary seasonally in an old-field climate change experiment. Glob. Change Biol. 18, 336e348. Tilman, D., 1999. The ecological consequences of changes in biodiversity: a search for general principles. Ecology 80, 1455e1474. Tilman, D., Downing, J.A., 1994. Biodiversity and stability in grasslands. Nature 367, 363e365. Tilman, D., Lehman, C.L., Thomson, K.T., 1997. Plant diversity and ecosystem productivity: theoretical considerations. Proc. Natl. Acad. Sci. U. S. A. 94, 1857e1861. Tilman, D., Reich, P.B., Isbell, F., 2012. Biodiversity impacts ecosystem productivity as much as resources, disturbance, or herbivory. Proc. Natl. Acad. Sci. U. S. A. 109, 10394e10397. Torsvik, V., Ovreas, L., Thingstad, T.F., 2002. Prokaryotic diversity - magnitude, dynamics, and controlling factors. Science 296, 1064e1066. Tuomi, P., Fagerbakke, K.M., Bratbak, G., Heldal, M., 1995. Nutritional enrichment of a microbial community: the effects on activity, elemental composition, community structure and virus production. FEMS Microbiol. Ecol. 16, 123e134. Unger, S., M aguas, C., Pereira, J.S., David, T.S., Werner, C., 2010. The influence of precipitation pulses on soil respiration e assessing the “Birch effect” by stable carbon isotopes. Soil Biol. Biochem. 42, 1800e1810. , D., Krist van Elsas, J.D., Chiurazzi, M., Mallon, C.A., Elhottova ufek, V., Salles, J.F., 2012. Microbial diversity determines the invasion of soil by a bacterial pathogen. Proc. Natl. Acad. Sci. U. S. A. 109, 1159e1164. Wagg, C., Bender, S.F., Widmer, F., van der Heijden, M.G.A., 2014. Soil biodiversity and soil community composition determine ecosystem multifunctionality. Proc. Natl. Acad. Sci. U. S. A. 111, 5266e5270. Wang, X., Liu, L., Piao, S., Janssens, I.A., Tang, J., Liu, W., Chi, Y., Wang, J., Xu, S., 2014. Soil respiration under climate warming: differential response of heterotrophic and autotrophic respiration. Glob. Change Biol. 20, 3229e3237. Wardle, D.A., Bardgett, R.D., Klironomos, J.N., Setala, H., van der Putten, W.H., Wall, D.H., 2004. Ecological linkages between aboveground and belowground biota. Science 304, 1629e1633. Wertz, S., Degrange, V., Prosser, J.I., Poly, F., Commeaux, C., Freitag, T., Guillaumaud, N., Roux, X.L., 2006. Maintenance of soil functioning following erosion of microbial diversity. Environ. Microbiol. 8, 2162e2169. Wertz, S., Degrange, V., Prosser, J.I., Poly, F., Commeaux, C., Guillaumaud, N., Le Roux, X., 2007. Decline of soil microbial diversity does not influence the resistance and resilience of key soil microbial functional groups following a model disturbance. Environ. Microbiol. 9, 2211e2219. Whitaker, J., Ostle, N., Nottingham, A.T., Ccahuana, A., Salinas, N., Bardgett, R.D., Meir, P., McNamara, N.P., 2014. Microbial community composition explains soil respiration responses to changing carbon inputs along an Andes-to-Amazon elevation gradient. J. Ecol. 102, 1058e1071. Yachi, S., Loreau, M., 1999. Biodiversity and ecosystem productivity in a fluctuating environment: the insurance hypothesis. Proc. Natl. Acad. Sci. U. S. A. 96, 1463e1468. Zhang, F.-G., Zhang, Q.-G., 2015. Patterns in species persistence and biomass production in soil microcosms recovering from a disturbance reject a neutral hypothesis for bacterial community assembly. PLoS ONE 10, e0126962. Zhou, J.Z., Xue, K., Xie, J.P., Deng, Y., Wu, L.Y., Cheng, X.H., Fei, S.F., Deng, S.P., He, Z.L., Van Nostrand, J.D., Luo, Y.Q., 2012. Microbial mediation of carbon-cycle feedbacks to climate warming. Nat. Clim. Change 2, 106e110. Zhou, X., Wan, S., Luo, Y., 2007. Source components and interannual variability of soil CO2 efflux under experimental warming and clipping in a grassland ecosystem. Glob. Change Biol. 13, 761e775.
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Microbial diversity limits soil heterotrophic respiration and mitigates the respiration response to moisture increase Fen-Guo Zhang a, Quan-Guo Zhang b, * a
College of Life Sciences, Shanxi Normal University, Linfen, Shanxi 041004, China State Key Laboratory of Earth Surface Processes and Resource Ecology and MOE Key Laboratory for Biodiversity Science and Ecological Engineering, Beijing Normal University, Beijing 100875, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 December 2015 Received in revised form 20 April 2016 Accepted 24 April 2016 Available online 29 April 2016
Decline in soil biodiversity associated with anthropogenic activities has raised concerns about the consequences for ecosystem functions. It remains uncertain how important soil microbial diversity is relative to abiotic factors, and how they interact, in driving ecosystem processes. Here we present results of a soil microcosm experiment in which microbial diversity and moisture conditions were independently manipulated. Loss of microbial diversity led to higher rates of soil microbial respiration, and the diversity effect was maintained over time during the course of the experiment. Higher moisture also enhanced soil respiration; but the moisture effect reduced over time, more rapidly in microcosms of higher microbial diversity. Overall, loss of microbial diversity enhanced soil respiration to a greater extent than moisture elevation, and also exacerbated the response of soil respiration to water addition. Loss of negative species interactions in microcosms of lower microbial diversity might be the major reason for the diversity effects observed in this study. Our results suggest that the integrity of soil microbial communities be crucial for the maintenance of soil carbon storage function. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Biodiversity Ecosystem functioning Global change Microbiome Soil respiration
1. Introduction Loss of species diversity may cause significant changes in ecosystem functions, with its effects being of comparable magnitude of that of many abiotic environmental factors including nutrient availability and temperature (Tilman, 1999; Loreau et al., 2001; Reich et al., 2001; Hooper et al., 2012; Tilman et al., 2012; Allison et al., 2013; Eisenhauer et al., 2013; Boyero et al., 2014; Bradford et al., 2014; Steinauer et al., 2014). Meanwhile, species diversity may interact with the abiotic factors as drivers of ecosystem processes. Species diversity may, on one hand, alleviate the impacts of stressful environmental factors such as drought (Tilman and Downing, 1994; Yachi and Loreau, 1999; Mulder et al., 2001; Gonzalez and Loreau, 2009; Awasthi et al., 2014), and on the other hand, enhance ecosystem responses to ‘positive’ perturbations including nutrient enrichment (Reich et al., 2001; He et al., 2002; Fridley, 2003). This can result from a ‘sampling effect’ as more diverse communities are more likely to contain particular
* Corresponding author. E-mail addresses: [email protected] (F.-G. Zhang), [email protected] (Q.-G. Zhang). http://dx.doi.org/10.1016/j.soilbio.2016.04.017 0038-0717/© 2016 Elsevier Ltd. All rights reserved.
species that are very resistant to environmental stress or highly responsive to improved conditions (Aarssen, 1997; Huston, 1997), or niche complementarity and facilitation among species because wider ranges of functional traits in species-richer communities can positively affect ecosystem performance under novel environmental conditions (Tilman et al., 1997; Loreau, 2000). Microbes, particularly those inhabiting topsoil, are characterized by tremendous diversity which is now threatened by anthropogenic activities including agricultural intensification and land use changes (Maeder et al., 2002; de Vries et al., 2013; Paula et al., 2014). While it seems reasonable to expect functional redundancy among hyper-diverse microbes (Torsvik et al., 2002; Nannipieri et al., 2003; Allison and Martiny, 2008; Cleveland et al., 2014), experimental studies that manipulated microbial communities often, although not always, found negative consequences of microbial diversity loss for ecological functions such as nitrogen cycling or biomass production (Degens, 1998; Wertz et al., 2007; Hol et al., 2010; Peter et al., 2011; van Elsas et al., 2012; Philippot et al., 2013; Wagg et al., 2014). However, it remains uncertain how important microbial diversity is relative to abiotic factors, and how they interact, in driving ecosystem processes (but see Degens, 1998; Wertz et al., 2007; Awasthi et al., 2014).
F.-G. Zhang, Q.-G. Zhang / Soil Biology & Biochemistry 98 (2016) 180e185
Here we experimentally address the dependency of soil microbial respiration on microbial diversity and soil moisture. Knowledge of the determinants of soil microbial respiration is crucial for a better understanding of global carbon cycles under future climatic conditions (Spehn et al., 2000; Curiel Yuste et al., 2007; Curiel Yuste et al., 2010; Suseela et al., 2012; Zhou et al., 2012; Li et al., 2013). There has been a rich literature on how abiotic factors, including temperature and moisture, affect soil respiration; however, microbial diversity and composition have often been confounding factors as they co-vary with the abiotic factors (Carney et al., 2007; Cleveland et al., 2007; Allison et al., 2013; Karhu et al., 2014; Matulich and Martiny, 2014; Whitaker et al., 2014; GarcíaPalacios et al., 2015). It is possible that higher species diversity allows microbial communities to achieve more reliable ecosystem functions under different moisture regimes. We carried out a microcosm experiment where microbial diversity and soil moisture were manipulated independently, to address their interaction in driving soil heterotrophic respiration.
2. Materials and methods 2.1. Soil collection and treatment Soil was collected from the top 15 cm of a semiarid grassland (Xilingol, Inner Mongolia, China; 43 320 N, 116 300 E), where Leymus chinensis was the dominant plant species. The soil was sieved to <2 mm, homogenized, and divided into microcosms of 160 g (equivalent dry mass) in 250 mL Schott Duran bottles. These microcosms were sterilized by 100 kGy gamma irradiation (Hongyisifang radiation technology Co., Ltd, Beijing, China), with soil sterility checked by enumeration of culturable bacteria on nutrient agar plates (3 g L1 beef extract, 10 g L1 peptone, 5 g L1 NaCl and 15 g L1 agar). Microcosms were then inoculated with suspensions of the same soil that had not been sterilized. Inocula were prepared by homogenizing 50 g of soil (equivalent dry mass) in 100 mL of sterile demineralized water by grinding and vortexing, followed by serial dilutions in sterile demineralized water. Sterile soil microcosms were inoculated with dilutions to create inocula equivalent to 102, 104, and 106 g of non-sterile soil per g sterile soil, 18 replicates per treatment level. Soil microcosms were incubated for recovery for 1 month at 25 C (which is near the mean topsoil temperature at the soil collection site in June and July), with bottle lids loosened and moisture content adjusted to 10% gravimetric water content (water/dry soil; near the moisture content of our source soil) regularly by addition of sterile water. Previous studies showed that the dilution disturbance could lead to a significant decrease in microbial diversity (Wertz et al., 2006; Peter et al., 2011; van Elsas et al., 2012). In the present study, experimental microcosms were incubated for recovery for one month, a period that is likely long enough for a full recovery in microbial activity (Zhang and Zhang, 2015). Analyses of soil property and microbial composition were carried out immediately after the 1-month recovery incubation (with six replicate microcosms randomly chosen from each level of dilution treatment for the measurements). Microcosms recovering from different levels of dilution treatments showed no detectable difference in total carbon or nitrogen content (Appendix A: Methods A1, Fig. A1). Pyrosequencing analyses of bacterial and fungal species composition (see detailed experimental procedures in Appendix A: Methods A2) confirmed that increasing levels of dilution treatments led to progressively decreased bacterial and fungal diversity (Fig. 1; Figs. A2 and A3). The microcosms under the 102, 104, and 106 dilution treatments are hereafter referred to as high-, intermediate- and low-diversity microcosms, respectively.
181
2.2. Experimental setup and measurement of soil respiration After the 1-month recovery growth, microcosms were incubated for another 46 days under three different moisture conditions. The 18 microcosms at each level of microbial diversity were randomly assigned into three groups, six replicates per group. Each group was exposed to one of the following three moisture treatments: low (10% gravimetric water content, near 30% of the waterholding capacity), high (20%), or fluctuating moisture (changing between 10% and 20%). For the low- or high-moisture microcosms, water content was maintained at 10% or 20% by adding sterile water at a 4-day interval (at day 1, 5, etc.). For the fluctuating-moisture microcosms, moisture content was increased to 20% at day 1. Then moisture was measured at a 4-day interval; when the average moisture of the microcosms became <10%, moisture of every microcosm was increased abruptly to 20% (pulse water addition was performed at day 1, 17 and 37; Fig. A4). Respiration rate of each microcosm was measured at a 4-day interval (at day 2, 6, etc.), with a Li-Cor 840A CO2/H2O analyzer (Li-Cor Environmental, Lincoln, NE, USA). Before measurement, each microcosm (bottle) was placed in water bath at 25 C and sealed with a rubber stopper. The rubber stopper had two ports that were connected with the gas analyzer, thus the bottle and the analyzer became a closed system. The analyzer maintained an air flow through this closed system, and recorded temperature, air pressure, CO2 and water content every second. For each microcosm, we ran the measurement for 70 s; data from 31 to 70 s were used for CO2 efflux calculation (fluctuations in air pressure and CO2 concentration may be observed at the beginning of the 70-s measurement, but then air pressure would remain constant and CO2 concentration increases gradually and linearly). The slope of CO2 concentration against time (dCO2/dt) was calculated, and the amount of CO2 evolved from the soil per second is the product of the slope and the volume of the system. The volume of the system was measured by injecting a known quantity of air with known CO2 concentration into the system that had been CO2-free (by flushing N2) and measuring the final CO2 concentration, with the dilution factor in CO2 concentration equal to the ratio of injected air volume to the system volume (Curiel Yuste et al., 2007). Soil respiration rate was expressed as mmol per g dry soil per second.
2.3. Data analysis There were three explanatory variables for soil respiration rates: level of microbial diversity, moisture treatment, and time. However, time may not be a meaningful explanatory variable for the fluctuating-moisture microcosms, as its effect can be confounded with that of moisture changes. To avoid this issue, we carried out two separate analyses. First, the average respiration rate over time was calculated for each microcosm, and analyzed using ANOVA, with diversity level and moisture treatment as two categorical explanatory variables. Second, respiration rates of the low- and high-moisture microcosms at all points in time were analyzed using linear mixed-effects model, with diversity level and moisture treatment as two categorical explanatory variables, time as a continuous explanatory variable, and microcosm ID as a random factor.
2.4. Accession numbers The pyrosequencing data have been deposited in NCBI Sequence Read Archive under accession number SRP057044.
182
F.-G. Zhang, Q.-G. Zhang / Soil Biology & Biochemistry 98 (2016) 180e185
Fig. 1. Species (OTUs) richness, Pielou's evenness and Shannon index of bacterial and fungal communities in a subset of experimental microcosms, after the 1-month recovery growth. Data show mean ± SE (n ¼ 6). Data points with the same letter within each panel do not significantly differ (Tukey multiple comparisons, Padj > 0.05). Fungal diversity indices in microcosms under 106 dilution treatment have been excluded from analysis, as in four out of the six microcosms PCR amplification for fungal communities was unsuccessful (note that a failure in amplification does not mean that the fungal abundance or diversity was zero).
3. Results 3.1. The effect of dilution treatments on soil microbial diversity A progressive decrease in microbial diversity with increasing magnitude of dilution treatment was observed, inferred by pyrosequencing analyses of bacterial and fungal communities in microcosms immediately after the 1-month recovery incubation. Compared with the microcosms of 102 dilution treatment, those under 104 treatment had a 35% decrease in bacterial species richness and a 31% decrease in fungal species richness, and those under 106 treatment had a 70% decrease in bacterial species richness (assays of fungal diversity failed; Fig. 1). 3.2. Overall effects of microbial diversity and moisture Analysis of the respiration rates averaged over time suggested a positive effect of microbial diversity loss on respiration (F2,45 ¼ 74.5, P < 0.001; Fig. 2d). Respiration rates of the intermediate- and lowdiversity microcosms were 48% and 61% higher than that of the
high-diversity microcosms, respectively. Soil respiration also showed a significant response to moisture content (F2,45 ¼ 15.3, P < 0.001), with high- and fluctuating-moisture treatments increasing respiration rates by 14% and 24%, respectively. There was a significant diversity moisture interaction effect (F4,45 ¼ 6.08, P < 0.001); high- and fluctuating-moisture treatments led to an increase in the rate of respiration in the intermediate- and lowdiversity microcosms, but not in the high-diversity microcosms (Fig. 2d). 3.3. Temporal changes in diversity and moisture effects Analysis of all repeated measures of respiration rates for the low- and high-moisture microcosms indicated significant effects of microbial diversity (F2,30 ¼ 54.2, P < 0.001), moisture treatment (low versus high moisture, F1,30 ¼ 15.6, P < 0.001) and diversity moisture interaction (F2,30 ¼ 13.6, P < 0.001; Fig. 2). Respiration rates did not show an overall trend over time (F1,389 ¼ 1.84, P ¼ 0.176). The effect of diversity time interaction was not significant (F2,389 ¼ 2.45, P ¼ 0.088), suggesting a persistent
F.-G. Zhang, Q.-G. Zhang / Soil Biology & Biochemistry 98 (2016) 180e185
183
lower microbial diversity (Fig. 2). 4. Discussion
Fig. 2. Respiration rates of soil microcosms under different diversity and moisture treatments. Both repeated measures throughout the experiment (aec) and averages over time (d) are shown. Within each diversity level in panel (d), bars with the same letter do not significantly differ (Tukey multiple comparisons, Padj > 0.05). Data show mean ± SE (n ¼ 6).
diversity effect on soil respiration over time. There was a significant moisture time interaction effect (F1,389 ¼ 78.8, P < 0.001), and a significant diversity moisture time interaction effect (F2,389 ¼ 3.72, P ¼ 0.025). Specifically, the high-moisture treatment enhanced respiration at the beginning of the experiment, regardless of microbial diversity level; then the moisture effect reduced over time, and the fade of this effect occurred very rapidly in the high-diversity microcosms and more slowly in microcosms of
Here we manipulated soil microbial diversity using a dilutionto-extinction approach. Microcosms recovering from different magnitudes of dilution treatments attained different levels of microbial diversity. We expected that a relatively short period (one month) of recovery incubation, on one hand, could allow a full recovery in microbial activity, and on the other hand, would not cause substantial difference in soil properties among microcosms of different levels of microbial diversity. The two assumptions have very likely held true. The fact that respiration rates of our microcosms during the main experiment did not show an overall trend over time suggests that the microbial activity had reached a stationary state during the recovery incubation; and the maintenance of diversity effect on soil respiration over time during the course of the main experiment implies that soil organic carbon content had not been decreased to a very low level during the recovery incubation to be the limiting factor for soil CO2 flux. Furthermore, we also found that our microcosms following the 1-month recovery incubation did not show significant difference in total carbon or nitrogen content among different microbial diversity treatments (Fig. A1). In our experiment, high microbial diversity and low moisture content represented more natural conditions. Both microbial diversity decline and moisture elevation caused higher rates of soil microbial respiration. The magnitude of the diversity effect on soil respiration was very large relative to the moisture effect. For instance, the intermediate-diversity microcosms, with a <40% decrease in bacterial or fungal species richness relative to the highdiversity microcosms (Fig. 1), had a ~48% increase in respiration rates (Fig. 2); the high-moisture treatments (with a 100% increase in moisture compared with the low-moisture treatment) increased respiration rates by only ~14% (Fig. 2). This is consistent with a previous suggestion that species diversity can show stronger effects on ecosystem processes than certain abiotic factors (Eisenhauer et al., 2013; Steinauer et al., 2014). Our finding that soil biodiversity limits soil respiration is consistent with a previous study (Bradford et al., 2014). We also found that loss of microbial diversity could exacerbate the responsiveness of soil respiration to moisture elevation. The high-diversity microcosms observed a rapid reduction over time in the enhancement effect of soil respiration by moisture elevation; in microcosms of lower microbial diversity, however, the moisture effect on soil respiration persisted longer during the experiment (Fig. 2). There are two possible explanations for the negative effect of microbial diversity on soil respiration. First, negative species interactions may limit decomposers' activity, and these species interactions may have been impaired in lower-diversity microcosms. One example is interference competition, as certain microbes may produce toxins that depress the growth of other species, or produce proteins that slow organic matter degradation by, e.g., contributing to micro-aggregate formation in soils (King, 2011). Trophic interactions may also negatively impact decomposers; for instance, top-down control of soil bacteria by viruses can slow soil heterotrophic respiration (Allen et al., 2010). Second, species-rich communities may contain particular species that had poor contributions to ecosystem functions but can somehow attain dominance. This is called ‘negative sampling effect’, which has been suggested to be common for non-biomass functions (Jiang et al., 2008). In the present study, microcosms exposed to higher dilution treatments may have shown a reduction in species diversity as well as a shift in species composition toward dominance by fastgrowing species. This is because the dilution treatment not only
184
F.-G. Zhang, Q.-G. Zhang / Soil Biology & Biochemistry 98 (2016) 180e185
removed species of very low abundances, but also decreased community sizes, thus species capable of rapid growth may have enjoyed a population growth advantage during the recovery incubation. The fast-growing species are likely to have higher respiration rates but lower carbon use efficiency. In particular, the abundances of fungal species involved in slower decomposition pathways (Wardle et al., 2004; Fierer et al., 2009; Strickland et al., 2009; de Graaff et al., 2010) may have decreased relative to bacteria in microcosms of lower microbial diversity (suggested by the fact that PCR assays of fungal communities have failed for low-diversity microcosms). Within bacteria, an increase in the abundances of Proteobacteria, and a decrease in Actinobacteria, were observed in lower-diversity microcosms (Fig. A2), the former being active decomposers, and the latter showing slow-growing behavior (Padmanabhan et al., 2003; Fierer et al., 2007). Either a negative sampling effect or negative species interactions may explain an overall negative diversity effect on soil respiration. For the interaction effect of microbial diversity and moisture, however, the negative species interactions hypothesis provide more likely explanations. Enhancement of decomposers' activity by improved moisture conditions can be depressed by a follow-up increase in the negative species interactions. For instance, moisture increase can immediately cause more active bacterial or fungal growth and thus increase the rate of soil respiration, but would also lead to further accumulation of proteins that limit decomposition, or an increase in the abundances of predators of the decomposers (Tuomi et al., 1995). The intermediate- and low-diversity microcosms may have lost a majority of the negative species interactions, allowing the enhancement effect by moisture elevation to persist longer. An explanation based on negative sampling effect appears less likely, which would be conditioned on (i) that functionally poor decomposers dominated high-diversity communities and (ii) that the poor decomposers also showed more rapid physiological acclimation to higher-moisture conditions. The fluctuating-moisture microcosms had insignificantly higher soil respiration rates relative to the high-moisture microcosms (Fig. 2) although the former had overall lower water content. Every episode of abrupt moisture elevation could stimulate soil respiration, maintaining a ‘Birch effect’, i.e., transient increase in CO2 flux following soil rewetting (Birch, 1964; Unger et al., 2010). This implies that the reduction of respiration rate over time in the highmoisture microcosms mainly results from biotic factors including negative species interaction, rather than resource or water limitation. It is possible that, in the fluctuating-moisture microcosms, factors slowing soil respiration during the high-moisture periods lost their effects when moisture became lower and the later pulse of moisture elevation could stimulate respiration once again. Our findings appear to contradict several recent studies that showed positive diversity effects on microbial processes (Wertz et al., 2007; Peter et al., 2011; van Elsas et al., 2012; de Vries et al., 2013; Philippot et al., 2013; Wagg et al., 2014). One possible explanation is that we measured a non-biomass function (CO2 flux) directly but some earlier studies actually measured biomass production functions or used biomass-based measurements to infer ecosystem processes (e.g. using copy number of certain genes to estimate the rate of nitrate oxidation), while biomass and nonbiomass functions may show different forms of dependence on species diversity (Jiang et al., 2008). The present study specifically suggests that loss of microbial diversity can increase the rate of soil microbial respiration and exacerbate the responsiveness of soil respiration to improved moisture conditions. It is of interest in future to investigate whether microbial diversity mediate the response of soil ecosystem functions to other types of environmental changes, in particular, labile carbon input that may lead to
priming effect on soil organic matter mineralization (Reinsch et al., 2013; Rousk et al., 2015). Our results imply that loss of microbial diversity may impair the soil carbon storage function under environmental changes that is currently a major concern (Carney et al., 2007; Cleveland et al., 2007; Zhou et al., 2007; Curiel Yuste et al., 2010; Suseela et al., 2012; Karhu et al., 2014; Wang et al., 2014). Therefore, among the management strategies for terrestrial carbon storage, those effectively protecting the integrity of soil microbial communities might be the most promising. Acknowledgements We thank O. Petchey for his helpful comments on the manuscript. This study was funded by the National Natural Science Foundation of China (31222010 and 31421063) and the 111 project (B13008). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2016.04.017. References Aarssen, L.W., 1997. High productivity in grassland ecosystems: effected by species diversity or productive species? Oikos 80, 183e184. Allen, B., Willner, D., Oechel, W.C., Lipson, D., 2010. Top-down control of microbial activity and biomass in an Arctic soil ecosystem. Environ. Microbiol. 12, 642e648. Allison, S.D., Lu, Y., Weihe, C., Goulden, M.L., Martiny, A.C., Treseder, K.K., Martiny, J.B.H., 2013. Microbial abundance and composition influence litter decomposition response to environmental change. Ecology 94, 714e725. Allison, S.D., Martiny, J.B.H., 2008. Resistance, resilience, and redundancy in microbial communities. Proc. Natl. Acad. Sci. U. S. A. 105, 11512e11519. Awasthi, A., Singh, M., Soni, S.K., Singh, R., Kalra, A., 2014. Biodiversity acts as insurance of productivity of bacterial communities under abiotic perturbations. ISME J. 8, 2445e2452. Birch, H.F., 1964. Mineralisation of plant nitrogen following alternate wet and dry conditions. Plant Soil 20, 43e49. Boyero, L., Cardinale, B.J., Bastian, M., Pearson, R.G., 2014. Biotic vs. abiotic control of decomposition: a comparison of the effects of simulated extinctions and changes in temperature. PLoS ONE 9, e87426. Bradford, M.A., Wood, S.A., Bardgett, R.D., Black, H.I.J., Bonkowski, M., Eggers, T., Grayston, S.J., Kandeler, E., Manning, P., Set€ al€ a, H., Jones, T.H., 2014. Discontinuity in the responses of ecosystem processes and multifunctionality to altered soil community composition. Proc. Natl. Acad. Sci. U. S. A. 111, 14478e14483. Carney, K.M., Hungate, B.A., Drake, B.G., Megonigal, J.P., 2007. Altered soil microbial community at elevated CO2 leads to loss of soil carbon. Proc. Natl. Acad. Sci. U. S. A. 104, 4990e4995. Cleveland, C.C., Nemergut, D.R., Schmidt, S.K., Townsend, A.R., 2007. Increases in soil respiration following labile carbon additions linked to rapid shifts in soil microbial community composition. Biogeochemistry 82, 229e240. Cleveland, C.C., Reed, S., Keller, A., Nemergut, D., O'Neill, S., Ostertag, R., Vitousek, P., 2014. Litter quality versus soil microbial community controls over decomposition: a quantitative analysis. Oecologia 174, 283e294. Curiel Yuste, J., Baldocchi, D.D., Gershenson, A., Goldstein, A., Misson, L., Wong, S., 2007. Microbial soil respiration and its dependency on carbon inputs, soil temperature and moisture. Glob. Change Biol. 13, 2018e2035. Curiel Yuste, J., Ma, S., Baldocchi, D.D., 2010. Plant-soil interactions and acclimation to temperature of microbial-mediated soil respiration may affect predictions of soil CO2 efflux. Biogeochemistry 98, 127e138. de Graaff, M.-A., Classen, A.T., Castro, H.F., Schadt, C.W., 2010. Labile soil carbon inputs mediate the soil microbial community composition and plant residue decomposition rates. New Phytol. 188, 1055e1064. bault, E., Liiri, M., Birkhofer, K., Tsiafouli, M.A., Bjørnlund, L., Bracht de Vries, F.T., The Jørgensen, H., Brady, M.V., Christensen, S., de Ruiter, P.C., d'Hertefeldt, T., Frouz, J., Hedlund, K., Hemerik, L., Hol, W.H.G., Hotes, S., Mortimer, S.R., Set€ al€ a, H., Sgardelis, S.P., Uteseny, K., van der Putten, W.H., Wolters, V., Bardgett, R.D., 2013. Soil food web properties explain ecosystem services across European land use systems. Proc. Natl. Acad. Sci. U. S. A. 110, 14296e14301. Degens, B.P., 1998. Decreases in microbial functional diversity do not result in corresponding changes in decomposition under different moisture conditions. Soil Biol. Biochem. 30, 1989e2000. Eisenhauer, N., Dobies, T., Cesarz, S., Hobbie, S.E., Meyer, R.J., Worm, K., Reich, P.B., 2013. Plant diversity effects on soil food webs are stronger than those of elevated CO2 and N deposition in a long-term grassland experiment. Proc. Natl. Acad. Sci. U. S. A. 110, 6889e6894.
F.-G. Zhang, Q.-G. Zhang / Soil Biology & Biochemistry 98 (2016) 180e185 Fierer, N., Bradford, M.A., Jackson, R.B., 2007. Toward an ecological classification of soil bacteira. Ecology 88, 1354e1364. Fierer, N., Strickland, M.S., Liptzin, D., Bradford, M.A., Cleveland, C.C., 2009. Global patterns in belowground communities. Ecol. Lett. 12, 1238e1249. Fridley, J.D., 2003. Diversity effects on production in different light and fertility environments: an experiment with communities of annual plants. J. Ecol. 91, 396e406. García-Palacios, P., Vandegehuchte, M.L., Shaw, E.A., Dam, M., Post, K.H., Ramirez, K.S., Sylvain, Z.A., de Tomasel, C.M., Wall, D.H., 2015. Are there links between responses of soil microbes and ecosystem functioning to elevated CO2, N deposition and warming? A global perspective. Glob. Change Biol. 21, 1590e1600. Gonzalez, A., Loreau, M., 2009. The causes and consequences of compensatory dynamics in ecological communities. Annu. Rev. Ecol. Evol. Syst. 40, 393e414. He, J.S., Bazzaz, F.A., Schmid, B., 2002. Interactive effects of diversity, nutrients and elevated CO2 on experimental plant communities. Oikos 97, 337e348. Hol, W.H.G., de Boer, W., Termorshuizen, A.J., Meyer, K.M., Schneider, J.H.M., van Dam, N.M., van Veen, J.A., van der Putten, W.H., 2010. Reduction of rare soil microbes modifies plant-herbivore interactions. Ecol. Lett. 13, 292e301. Hooper, D.U., Adair, E.C., Cardinale, B.J., Byrnes, J.E.K., Hungate, B.A., Matulich, K.L., Gonzalez, A., Duffy, J.E., Gamfeldt, L., O'Connor, M.I., 2012. A global synthesis reveals biodiversity loss as a major driver of ecosystem change. Nature 486, 105e108. Huston, M.A., 1997. Hidden treatments in ecological experiments: re-evaluating the ecosystem function of biodiversity. Oecologia 110, 449e460. Jiang, L., Pu, Z., Nemergut, D.R., 2008. On the importance of the negative selection effect for the relationship between biodiversity and ecosystem functioning. Oikos 117, 488e493. Karhu, K., Auffret, M.D., Dungait, J.A.J., Hopkins, D.W., Prosser, J.I., Singh, B.K., Subke, J.-A., Wookey, P.A., Agren, G.I., Sebastia, M.-T., Gouriveau, F., Bergkvist, G., Meir, P., Nottingham, A.T., Salinas, N., Hartley, I.P., 2014. Temperature sensitivity of soil respiration rates enhanced by microbial community response. Nature 513, 81e84. King, G.M., 2011. Enhancing soil carbon storage for carbon remediation: potential contributions and constraints by microbes. Trends Microbiol. 19, 75e84. Li, D., Zhou, X., Wu, L., Zhou, J., Luo, Y., 2013. Contrasting responses of heterotrophic and autotrophic respiration to experimental warming in a winter annualdominated prairie. Glob. Change Biol. 19, 3553e3564. Loreau, M., 2000. Biodiversity and ecosystem functioning: recent theoretical advances. Oikos 91, 3e17. Loreau, M., Naeem, S., Inchausti, P., Bengtsson, J., Grime, J.P., Hector, A., Hooper, D.U., Huston, M.A., Raffaelli, D., Schmid, B., Tilman, D., Wardle, D.A., 2001. Biodiversity and ecosystem functioning: current knowledge and future challenges. Science 294, 804e808. Maeder, P., Fliessbach, A., Dubois, D., Gunst, L., Fried, P., Niggli, U., 2002. Soil fertility and biodiversity in organic farming. Science 296, 1694e1697. Matulich, K.L., Martiny, J.B.H., 2014. Microbial composition alters the response of litter decomposition to environmental change. Ecology 96, 154e163. Mulder, C.P.H., Uliassi, D.D., Doak, D.F., 2001. Physical stress and diversityproductivity relationships: the role of positive interactions. Proc. Natl. Acad. Sci. U. S. A. 98, 6704e6708. Nannipieri, P., Ascher, J., Ceccherini, M.T., Landi, L., Pietramellara, G., Renella, G., 2003. Microbial diversity and soil functions. Eur. J. Soil Sci. 54, 655e670. Padmanabhan, P., Padmanabhan, S., DeRito, C., Gray, A., Gannon, D., Snape, J.R., Tsai, C.S., Park, W., Jeon, C., Madsen, E.L., 2003. Respiration of 13C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of C-13labeled soil DNA. Appl. Environ. Microbiol. 69, 1614e1622. Paula, F.S., Rodrigues, J.L.M., Zhou, J., Wu, L., Mueller, R.C., Mirza, B.S., Bohannan, B.J.M., Nüsslein, K., Deng, Y., Tiedje, J.M., Pellizari, V.H., 2014. Land use change alters functional gene diversity, composition and abundance in Amazon forest soil microbial communities. Mol. Ecol. 23, 2988e2999. Peter, H., Beier, S., Bertilsson, S., Lindstrom, E.S., Langenheder, S., Tranvik, L.J., 2011. Function-specific response to depletion of microbial diversity. ISME J. 5, 351e361. Philippot, L., Spor, A., Henault, C., Bru, D., Bizouard, F., Jones, C.M., Sarr, A., Maron, P.A., 2013. Loss in microbial diversity affects nitrogen cycling in soil. ISME J. 7, 1609e1619. Reich, P.B., Knops, J., Tilman, D., Craine, J., Ellsworth, D., Tjoelker, M., Lee, T., Wedin, D., Naeem, S., Bahauddin, D., Hendrey, G., Jose, S., Wrage, K., Goth, J., Bengston, W., 2001. Plant diversity enhances ecosystem responses to elevated CO2 and nitrogen deposition. Nature 410, 809e812. Reinsch, S., Ambus, P., Thornton, B., Paterson, E., 2013. Impact of future climatic
185
conditions on the potential for soil organic matter priming. Soil Biol. Biochem. 65, 133e140. Rousk, J., Hill, P.W., Jones, D.L., 2015. Priming of the decomposition of ageing soil organic matter: concentration dependence and microbial control. Funct. Ecol. 29, 285e296. Spehn, E.M., Joshi, J., Schmid, B., Alphei, J., Korner, C., 2000. Plant diversity effects on soil heterotrophic activity in experimental grassland ecosystems. Plant Soil 224, 217e230. Steinauer, K., Tilman, D., Wragg, P.D., Cesarz, S., Cowles, J.M., Pritsch, K., Reich, P.B., Weisser, W.W., Eisenhauer, N., 2014. Plant diversity effects on soil microbial functions and enzymes are stronger than warming in a grassland experiment. Ecology 96, 99e112. Strickland, M.S., Osburn, E., Lauber, C., Fierer, N., Bradford, M.A., 2009. Litter quality is in the eye of the beholder: initial decomposition rates as a function of inoculum characteristics. Funct. Ecol. 23, 627e636. Suseela, V., Conant, R.T., Wallenstein, M.D., Dukes, J.S., 2012. Effects of soil moisture on the temperature sensitivity of heterotrophic respiration vary seasonally in an old-field climate change experiment. Glob. Change Biol. 18, 336e348. Tilman, D., 1999. The ecological consequences of changes in biodiversity: a search for general principles. Ecology 80, 1455e1474. Tilman, D., Downing, J.A., 1994. Biodiversity and stability in grasslands. Nature 367, 363e365. Tilman, D., Lehman, C.L., Thomson, K.T., 1997. Plant diversity and ecosystem productivity: theoretical considerations. Proc. Natl. Acad. Sci. U. S. A. 94, 1857e1861. Tilman, D., Reich, P.B., Isbell, F., 2012. Biodiversity impacts ecosystem productivity as much as resources, disturbance, or herbivory. Proc. Natl. Acad. Sci. U. S. A. 109, 10394e10397. Torsvik, V., Ovreas, L., Thingstad, T.F., 2002. Prokaryotic diversity - magnitude, dynamics, and controlling factors. Science 296, 1064e1066. Tuomi, P., Fagerbakke, K.M., Bratbak, G., Heldal, M., 1995. Nutritional enrichment of a microbial community: the effects on activity, elemental composition, community structure and virus production. FEMS Microbiol. Ecol. 16, 123e134. Unger, S., M aguas, C., Pereira, J.S., David, T.S., Werner, C., 2010. The influence of precipitation pulses on soil respiration e assessing the “Birch effect” by stable carbon isotopes. Soil Biol. Biochem. 42, 1800e1810. , D., Krist van Elsas, J.D., Chiurazzi, M., Mallon, C.A., Elhottova ufek, V., Salles, J.F., 2012. Microbial diversity determines the invasion of soil by a bacterial pathogen. Proc. Natl. Acad. Sci. U. S. A. 109, 1159e1164. Wagg, C., Bender, S.F., Widmer, F., van der Heijden, M.G.A., 2014. Soil biodiversity and soil community composition determine ecosystem multifunctionality. Proc. Natl. Acad. Sci. U. S. A. 111, 5266e5270. Wang, X., Liu, L., Piao, S., Janssens, I.A., Tang, J., Liu, W., Chi, Y., Wang, J., Xu, S., 2014. Soil respiration under climate warming: differential response of heterotrophic and autotrophic respiration. Glob. Change Biol. 20, 3229e3237. Wardle, D.A., Bardgett, R.D., Klironomos, J.N., Setala, H., van der Putten, W.H., Wall, D.H., 2004. Ecological linkages between aboveground and belowground biota. Science 304, 1629e1633. Wertz, S., Degrange, V., Prosser, J.I., Poly, F., Commeaux, C., Freitag, T., Guillaumaud, N., Roux, X.L., 2006. Maintenance of soil functioning following erosion of microbial diversity. Environ. Microbiol. 8, 2162e2169. Wertz, S., Degrange, V., Prosser, J.I., Poly, F., Commeaux, C., Guillaumaud, N., Le Roux, X., 2007. Decline of soil microbial diversity does not influence the resistance and resilience of key soil microbial functional groups following a model disturbance. Environ. Microbiol. 9, 2211e2219. Whitaker, J., Ostle, N., Nottingham, A.T., Ccahuana, A., Salinas, N., Bardgett, R.D., Meir, P., McNamara, N.P., 2014. Microbial community composition explains soil respiration responses to changing carbon inputs along an Andes-to-Amazon elevation gradient. J. Ecol. 102, 1058e1071. Yachi, S., Loreau, M., 1999. Biodiversity and ecosystem productivity in a fluctuating environment: the insurance hypothesis. Proc. Natl. Acad. Sci. U. S. A. 96, 1463e1468. Zhang, F.-G., Zhang, Q.-G., 2015. Patterns in species persistence and biomass production in soil microcosms recovering from a disturbance reject a neutral hypothesis for bacterial community assembly. PLoS ONE 10, e0126962. Zhou, J.Z., Xue, K., Xie, J.P., Deng, Y., Wu, L.Y., Cheng, X.H., Fei, S.F., Deng, S.P., He, Z.L., Van Nostrand, J.D., Luo, Y.Q., 2012. Microbial mediation of carbon-cycle feedbacks to climate warming. Nat. Clim. Change 2, 106e110. Zhou, X., Wan, S., Luo, Y., 2007. Source components and interannual variability of soil CO2 efflux under experimental warming and clipping in a grassland ecosystem. Glob. Change Biol. 13, 761e775.