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For all enzymes measured, we found support for resource allocation theory as applied to soil mi- crobes. The decline in phosphatase activity with P additions is consistent with previous studies that P additions suppressed phosphatase activity (Clarholm, 1993;Olander and Vitousek, 2000). In our experiment, the likely cause is an increase in P availability relative to N availability, which increased microbial N demand relative to P. The shift toward higher N:P enzyme ratios with added P speaks to a shift in microbial resource allocation. In the complex N+P treatment, one might expect that in- creased N availability would maintain high microbial P demand and phosphatase activity. However,

complex forms of N require enzymatic degradation, while the supplemented P was in a labile form. It is, therefore, likely that P availability increased faster than N availability in this treatment, thus in- creasing microbial N demand more than P. This increased N demand was potentially alleviated over time as N availability increased. In the simple N+P treatment, extractable N accumulated rapidly in the soil, and phosphatase activity remained high, indicating that additions of labile N maintained microbial P demand. That the simple N treatment also contained excess P may explain why phos- phatase activity did not, in fact, increase relative to control soils; by providing soil microbes with these two nutrients in immediately assimilable forms, we may have avoided inducing P-limitation. The three C-degrading enzymes measured in this study (beta glucosidase, beta xylosidase and cel- lobiohydrolase) all behaved similarly in response to nutrients, increasing over time in response to simple N+P. High N availability, as indicated by high extractable N in this treatment, may have led to greater incorporation of N into microbial biomass, thus leading to the observed increase in organic N. Greater microbial biomass production can exacerbate microbial C-limitation, leading to the production of more C-acquiring enzymes, as has been observed in deciduous forests (Waldrop

et al., 2004;Sinsabaugh et al., 2005) and grasslands (Henry et al., 2005). In the complex N treat-

ment, reduced beta xylosidase and cellobiohydrolase activity coupled with decreased C:N enzyme ratios suggests microbial allocation towards increased N acquisition and decreased C acquisition. However, given that our complex N treatment also contained added C, it is possible that declines in beta xylosidase and cellobiohydrolase activity in this treatment represent a shift towards acquisition of the added protein substrate that is independent of changes in microbial stoichiometric demand. The one N-acquiring enzyme measured in this study (N-acetyl glucosaminidase) also responded to nutrient additions in a manner consistent with microbial resource allocation theory. The large in- crease inN-acetyl glucosaminidase activity in the complex N+P treatment suggests this enzyme’s production was strongly induced in response to complex N. The decline in C:N and increase in N:P enzyme ratios support the notion of a shift towards N-acquisition in the complex N treatment. The specific function of this enzyme is the hydrolysis of β-N-acetylglucosamine residues from oligosaccharides (German et al., 2011). WhileN-acetyl glucosaminidase is considered functionally

important in soils as a chitin or peptidoglycan-degrading enzyme, it is also involved in the degly- cosylation of some proteins (Stals et al., 2010). Our finding that this common soil enzyme appears inducible by intermediate degradation products that signal substrate availability is consistent with our understanding of microbial physiology (Priest, 1977) and with previous nutrient manipulation experiments in soils (Allison and Vitousek, 2005;Averill and Finzi, 2011). It should also be noted that the increase inN-acetyl glucosaminidase activity over time in the simple N+P treatment may speak to the dual role of this enzyme as a C and N-acquiring enzyme. All sources of organic N also contain C, and chitin, as the second most abundant biopolymer on earth (Jolles and Muzzarelli, 1999), is considered both an important C and N source for microorganisms.

The second objective of this study was to evaluate the role of roots in mediating enzymatic responses to nutrients. We hypothesized that in the complex N treatment, root-derived C in the form of in- creased root biomass or exudates would alleviate microbial C limitation and lead to greater enzyme activity, while additions of simple N would have the reverse effect. We found support for the first part of this hypothesis, in that mean root biomass in the complex N+P treatment was greater than controls and mean root biomass in the simple N+P treatment was less than controls. The fact that the largest increase in root biomass occurred in the P-only treatment may speak to the fact that P is a more limiting nutrient than N in these soils, as has been widely observed across tropical forests (Vitousek et al., 2010). Additionally, the large increase in shoot biomass in the complex N relative to all other treatments suggests that seedling growth was substantially enhanced by microbial degra- dation of organic N. However, these shifting allocation patterns in plants did not appear to influence microbial enzyme activity very strongly in most cases, and in no instances did we find the antici- pated planting×nutrient effect in response to complex N. There are several potential explanations for the lack of a strong effect of roots on enzyme activities. Firstly, our hypothesis that enzyme activity would be enhanced in the presence of roots is premised on the assumption that microbial biomass is C-limited, however, microbial biomass is often found to be P limited (Cleveland and Liptzin, 2007), particularly in tropical soils (Cleveland and Townsend, 2006). If microbial biomass were N or P limited, microbial responses to root-derived C additions would be constrained by N or P availability, as demonstrated in recent modeling studies (Drake et al., 2013) and in studies testing

nutrient co-limitation of microbial activity (e.g., Vance and Chapin (2001); Brown et al. (2009);

Krashevska et al.(2010)). In our simple N treatment, increases in C-acquiring enzyme activity over

time indicate a shift towards microbial C-limitation, however, we were unable to determine whether roots had a positive effect on enzyme activities following repeated mineral N additions due to the death of most seedlings by the end of this treatment. Another possibility suggested by seedling death in the simple N treatment is that our N addition rate was high compared to T. heterophylla N requirements and that root priming effects were consequently reduced in N treatments. This is also consistent with the fact that we did not observe significant differences in extractable N be- tween planted and unplanted soils, despite the increased incorporation of N into plant biomass as evidenced by the dramatically lower root and shoot C:N ratios in the N treatments compared with controls. Additionally, our sterilization ofT. heterophyllaseeds and subsequent germination under sterile conditions could have decreased mycorrhizal fungal colonization, which can substantially af- fect plant allocation patterns (Rygiewicz and Andersen, 1994). Finally, in the complex N treatment microbes may not have had to rely heavily on root exudates as a source of labile C due to the fact that casein also contains C that can be released through enzymatic degradation.

We recognize that a lack of microbial biomass measurements limits our interpretations with respect to microbial resource stoichiometry. Changes in enzyme activities can represent both shifts in total activity and shifts in activity per unit microbial biomass (specific activity), and differences in total enzyme activity can therefore be confounded with differences in the size of the microbial population. For instance, additions of mineral N fertilizer are often found to reduce microbial biomass (Compton

et al., 2004; Treseder, 2008; Wallenstein et al., 2006), and reductions in enzyme activity in N-

amended soils may therefore represent a decline in the size of the microbial biomass rather than a shift in resource allocation. Examining ratios of C:N, C:P and N:P acquiring enzyme activity provided us with an alterative method for evaluating nutrient demand in the absence of biomass data. However, enzymatic C:N:P ratios are generally found to converge on 1:1:1, suggesting that the plasticity of these ratios is relatively constrained (Sinsabaugh et al., 2008, 2009). We also acknowledge limitations associated with extrapolating from laboratory enzyme assays, which can provide an index of potential enzyme activity, to activityin situ, which can be hindered by diffusion

limitation, enzyme-mineral interactions, variable substrate concentrations and other physical and chemical properties of the soil matrix (Wallenstein and Weintraub, 2008;German et al., 2011). Although the changes in enzyme activities we observed are consistent with resource allocation the- ory, there are alternative interpretations to our results that speak to the complexity of the interactions both within soil microbial communities and with their environment. Microbial community structure often shifts in response to nutrients (e.g.,Frey et al.(2004);Allison et al.(2007);Allison and Mar- tiny(2008);Nemergut et al.(2008)). Changes in community structure can occur due to changes in the relative fitness of various microorganisms under altered nutrient regimes (Fierer and Jackson, 2006;Allison et al., 2007;Fierer et al., 2007), leading to selection for organisms that can better ex- ploit available resources. Such community shifts could alter the types of and abundances of enzyme producers. Shifts in bacterial:fungal ratios, which are often observed in N-addition studies, can alter the stoichiometry of the microbial biomass, which in turn can affect resource demand as well as the types of enzymes produced. N additions can also directly inhibit the production of some enzymes by soil fungi (Fog, 1988;Carreiro et al., 2000;Saiya-Cork et al., 2002).