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C and N cycles

Plant species differed in their effects on attributes of N cycle when they showed an effect on nitrate and net nitrification while ammonium and net ammonification was not impacted. L. perenne and P.

trivilialis showed higher soil nitrate than A. odoratum, F. rubra and H. lanatus, and also non-

significantly higher values for net nitrification than the other plant species. These results would suggest an increase of rate of N cycling in the soil of these species. Higher level of potential N mineralization was found for L. perenne in comparison to A. odoratum in a mesocosm study by De Deyn et al. (2012). Harrison and Bardgett (2010) also found differences in effect of species on soil N attributes and the difference was between legumes and species from other functional groups tested. However, they did not find differences in nitrate among F. rubra, A. odoratum and L. perenne while difference between L. perenne and F. rubra was found in the present study. Their study was only a short term in contrary to the present study spanning two growing seasons. This may point towards a need for longer time plants to develop soil feedbacks resulting from inherent plant strategies (Orwin

et al., 2010).

The present results also suggested an effect of plant life strategy on soil N cycle which was shown by higher soil nitrate and net nitrification for fast growing plant species in comparison to slow growing species, in accordance with hypothesis 1. This agrees with theory and studies showing relevance of plant leaf economic spectrum (Wright et al., 2004) for dynamics of soil N cycle (Orwin et al., 2010; Grigulis et al., 2013; Kastovska et al., 2014) whereby fast growing plant species (as determined by high leaf N and low leaf C:N) are associated with greater soil N availability. Orwin et al. (2010) showed that grass species with high leaf and litter N content grown in monoculture plots for 7 years were associated with higher soil inorganic N pool and to a lesser extent net N mineralization and nitrification. In grassland multisite comparison study, leaf N content showed a positive correlation with litter decomposition rate in two out of three sites (Grigulis et al., 2013) suggesting greater release of nutrients into the soil for more nutrient exploitative plants. They further argued that more exploitative plat strategies are expected to result in greater plant biomass but poor C and nutrient

169 retention as they are associated with microbial communities performing rapid rates of

mineralization and nitrification. On the other hand, potential nitrification rate was not associated with any of plant traits measured (e.g. leaf N and C:N ratio) but solely with availability of soil ammonium (Legay et al., 2014) suggesting that plant effects on soil N cycling might be indirect. Similarly, Grigulis et al. (2013) found that soil processes related to N cycling (e.g. potential N mineralization) were largely explained by microbial parameters and not plant traits.

Greater soil N availability is generally considered to result from decomposition of high litter quality (low C:N ratio) however, aboveground litter was removed in the present study, and while root decomposition effect cannot be discounted, it can be anticipated that different plant life strategies will affect soil N cycle through their association with soil microbial community. For instance, Orwin et al. (2010) showed that fast growing plants were associated with higher bacterial:fungi ratio. Bacterial food web was found to be associated with greater soil N cycling in a large-scale survey (De Vries et al., 2013). Plants through rhizodeposition release a substantial portion of primary

productivity (Pasch and Kuzyakov, 2017; Hirte et al., 2018) and its quantity as well as quantity would affect microbial community structure and activity (Bardgett and Wardle, 2010) and microbial community attributes were found strong drivers of soil N cycle (Grigulis et al., 2013). Legay et al. (2014) hypothesized that root C:N ratio might influence exudate C:N ratio and labile organic compounds C:N ratio which might affect soil denitrification. The mechanism for higher soil N availability for fast growing plant species may thus be related to generally expected higher rhizodeposition by fast growing species, which have been also shown by Kastovska et al. (2014), when the increased rhizodeposition may promote N mineralization in the soil (Dijkstra et al., 2013). However, it was also shown, that the effect of plant life strategy was strongly supported by only two fast growing species but not by H. lanatus which did not differ from both slow growing species. This may suggest that life strategy concept is a continuum of plant species allocation in a two-

dimensional space than a strict separate grouping into fast- and slow- growing plants. Alternatively, N cycle attributes related to soil mineral N and net mineralization rates in the peak season may not fully capture the dynamics of N transformations and availability (Schimel and Bennet, 2004). N demand of plants have been shown to differ temporarily during season with the highest plant demand in spring which then decreased towards autumn (Kastovska et al., 2014) thus the higher levels of soil N in the present study may simply reflect seasonal changes due to plant phenology. Interestingly, bare soil mesocosms had higher nitrate compared to most of species but P. trivialis. The high soil nitrate concentration might have reflected that the soil for the mesocosms was taken from intact grassland and N transformation processes may have continued without plant presence in until the following season and energy source for microbial community was provided from root decomposition. Disturbance related to soil processing during mesocosms set up may have made these roots available for decomposition. Lack of plant N demand would then result in accumulation of nitrate. On the other hand, highest soil nitrate concentration for bare soil than for most plant species and differences in soil nitrate among plant species might also suggest differential plant nitrate uptake between the species. This would also mean lower N pool in plant aboveground biomass for P. trivialis which showed similar nitrate as bare soil and higher than the other species. However, this was only true for F. rubra while the other species had similar N pool in aboveground biomass as P. trivialis. Nevertheless, the differences might be present for belowground biomass N pool which was not determined.

Aboveground biomass C:N ratio was affected by plant species and plant life strategy whereby it was higher for A. odoratum in comparison to P. trivialis and it was higher for slow growing plant species compared to fast growing species. It confirms that evolutionary trade-offs in plat growth strategies results in differences in biomass nutrients typically assessed by leaf characteristics (Wright et al.

170 2004) but assessed as whole aboveground plant biomass characteristics in the present study. Plant biomass C and N stoichiometry reflects relative strength of plant C and N metabolism (Luo et al., 2017). It can affect decomposer food web in the soil through differences in response of soil fungal and bacterial communities to plant litter of different C:N ratio as shown by (Rousk and Bååth, 2007) whereby fungal growth was promoted more by substrate with higher C:N than lower C:N and bacteria showed an opposite response. Furthermore, changes in soil microbial food web dominance can have consequences for ecosystem processes when fungal dominated food web has been linked to conservative nutrient cycling (De Vries et al., 2011; De Vries et al., 2013) and is expected to promote soil C storage (J. Six et al., 2006). On the other hand, C:N litter ratio as a measure of litter quality can affect fate of litter degradation in the soil and differentially contribute to SOC storage whereby higher quality litter might be more efficiently assimilated into microbial biomass resulting in a greater microbial biomass and microbial by-products available for stabilization (Cotrufo et al., 2013).

Species did not differ in ecosystem respiration at any time point tested and overall effects on the respiration measured in the three time points during plant growth towards peak of season were only due to higher ecosystem respiration for plant species mesocosms in comparison to bare soil

mesocosms. Similarity in ecosystem respiration between grass species of A. odoratum and L.

perenne grown in monocultures have been shown by others (Orwin et al., 2010; De Deyn et al.,

2012). On the other hand, differences in ecosystem respiration between grassland species were found when different functional groups were compared. Legumes showed a higher respiration in comparison to grasses and forbs (Orwin et al., 2010; De Deyn et al., 2012). But also differences within C3 grasses have been shown by (Orwin et al., 2010) comparing Festuca ovina to L. perenne. They showed that F. ovina was weakly associated with higher root biomass and with high microbial biomass C and biomass which could have been behind the positive effect on ecosystem respiration rates. This suggests that rates of C cycling are unlikely to be predictable based solely on leaf and litter quality (Orwin et al., 2010).

Microbial community response in monocultures

Data suggested that response of bacterial community structure was primarily related to differences between bare soil and planted mesocosms. These differences were shown to be associated with changes of relative abundance of microbial taxa associated with plant roots such as

Alphaproteobacteria and Acidobacteria by Thompson et al. (2013). However, relative abundances of Alphaproteobacteria (ANOVA, F5,13 = 4.3, Padjusted = 0.24) and Acidobacteria were not affected in the present study. Similarly, plant life strategies would show different effect on soil bacterial

communities as they shown an effect on soil N availability. They would also promote copiotrophic bacterial taxa due to anticipated differences in exudation pattern between fast and slow growing plant species (Kastovska et al., 2014). Nevertheless, this was not suggested by the present data. Only orders Opiputalles (Opitutae) and Rickettsiales (Alphaproteobacetria) showed responses. Opiputalles responded to plant species. Hester et al. (2018) found this order to be positively affected by N addition treatment and it was also weakly correlated with N2O emissions. Its members were described as polysaccharide-utilizing bacteria capable of nitrate reduction to nitrite (Chin et al., 2001). They have been found associated with rhizosphere of diverse plants such as sugarcane and wetland plants (Soil et al., 2018). Order Rickettsiales showed a response to both plant species and plant life strategy. Members of Rickettsiales are mainly associated with arthropods hosts but were also found as algae endosymbionts and cannot survive in the long term outside of their host (Kawafune et al., 2012), nevertheless, they are not typically abundant in the soil.

171 Lower overall response of bacterial community to plant species in monocultures can be due to relatively low impact of plant species on soil nutrient level in the mesocosms. Despite change of soil N cycle attributes between fast growing and slow growing plants were found, the overall quality and quantity of soil organic matter may have been unaffected during the relatively short duration of the experiment. As such, bacterial response to these relatively small changes may have been overruled by similarity of organic matter characteristics in the mesocosms of different plant species (Millard and Singh, 2010). Moreover, only transient changes in relative abundance may have been masked by presence of relic DNA (Carini et al., 2016).

The fungal community showed changes in both community structure and relative abundance of fungal taxa at order and individual ASVs resolution. Changes in fungal community with plant alteration can be expected as close association of the fungal community with the plant community have been shown before (e.g. Cassman et al., 2016) and can be related to plant-fungal relationship in the form of mutualistic or pathogenic associations (Putten et al., 2013) or to plant litter driven fungal saprotrophs (Millard and Singh, 2010). Indeed, indicator species analysis showed that true pathotrophs increased their relative abundance within taxa associated only with single plant species.