Plant Diversity Surpasses Plant Functional Groups and Plant Productivity as Driver of Soil Biota in the Long Term Nico Eisenhauer1*, Alexandru Milcu2, Alexander C. W. Sabais3, Holger Bessler4, Johanna Brenner5, Christof Engels4, Bernhard Klarner5, Mark Maraun5, Stephan Partsch3, Christiane Roscher6, Felix Schonert3, Vicky M. Temperton7, Karolin Thomisch5, Alexandra Weigelt8, Wolfgang W. Weisser9, Stefan Scheu5 1 Department of Forest Resources, University of Minnesota, St. Paul, Minnesota, United States of America, 2 Division of Biology, NERC Centre for Population Biology, Imperial College London, Ascot, United Kingdom, 3 Institute of Zoology, Darmstadt University of Technology, Darmstadt, Germany, 4 Department of Plant Nutrition and Fertilization, Humboldt University of Berlin, Berlin, Germany, 5 J.F. Blumenbach Institute of Zoology and Anthropology, Georg-August-University Goettingen, Goettingen, Germany, 6 Department of Community Ecology, Helmholtz Centre for Environmental Research - UFZ, Halle, Germany, 7 IBG-2 Plant Sciences, Forschungszentrum Juelich GmbH, Juelich, Germany, 8 Department of Botany and Functional Biodiversity, Institute of Biology I, University of Leipzig, Leipzig, Germany, 9 Institute of Ecology, Friedrich-Schiller-University Jena, Jena, Germany Abstract Background: One of the most significant consequences of contemporary global change is the rapid decline of biodiversity in many ecosystems. Knowledge of the consequences of biodiversity loss in terrestrial ecosystems is largely restricted to single ecosystem functions. Impacts of key plant functional groups on soil biota are considered to be more important than those of plant diversity however, current knowledge mainly relies on short-term experiments. Methodology/Principal Findings: We studied changes in the impacts of plant diversity and presence of key functional groups on soil biota by investigating the performance of soil microorganisms and soil fauna two, four and six years after the establishment of model grasslands. The results indicate that temporal changes of plant community effects depend on the trophic affiliation of soil animals: plant diversity effects on decomposers only occurred after six years, changed little in herbivores, but occurred in predators after two years. The results suggest that plant diversity, in terms of species and functional group richness, is the most important plant community property affecting soil biota, exceeding the relevance of plant above- and belowground productivity and the presence of key plant functional groups, i.e. grasses and legumes, with the relevance of the latter decreasing in time. Conclusions/Significance: Plant diversity effects on biota are not only due to the presence of key plant functional groups or plant productivity highlighting the importance of diverse and high-quality plant derived resources, and supporting the validity of the singular hypothesis for soil biota. Our results demonstrate that in the long term plant diversity essentially drives the performance of soil biota questioning the paradigm that belowground communities are not affected by plant diversity and reinforcing the importance of biodiversity for ecosystem functioning. Citation: Eisenhauer N, Milcu A, Sabais ACW, Bessler H, Brenner J, et al. (2011) Plant Diversity Surpasses Plant Functional Groups and Plant Productivity as Driver of Soil Biota in the Long Term. PLoS ONE 6(1): e16055. doi:10.1371/journal.pone.0016055 Editor: Andrew Hector, University of Zurich, Switzerland Received September 2, 2010 Accepted December 6, 2010 Published January 7, 2011 Copyright: �� 2011 Eisenhauer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: German Science Foundation (FOR 456), German Science Foundation (Ei 862/1-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nico.eisenhauer@web.de Introduction Mankind faces multiple anthropogenic global environmental changes, which are now large enough to exceed the bounds of natural variability [1,2]. One of the most significant consequences of contemporary global change is the rapid decline of biodiversity in many ecosystems [3���6]. This unprecedented biodiversity loss has generated concern over the consequences for ecosystem functioning and services, and prompted a multitude of studies [7,8]. Most studies focused on the effects of diversity loss on single trophic levels or measures of ecosystem functioning, considerably less attention has been paid to the consequences of plant diversity loss for the performance of multiple trophic levels and ecosystem functions [8���11]. This is surprising since cascading effects of biodiversity loss may result in a vicious circle of diversity loss due to the interconnectance between ecosystem or foodweb compo- nents, and the incidence of soil feedback mechanisms [12���14]. Terrestrial grasslands are widespread model systems for investigating the biodiversity ��� ecosystem functioning relationship [7,15���18]. Soil biota constitute a significant component of terrestrial ecosystems by governing essential ecosystem functions, such as decomposition and recycling of organic residues, and PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16055

thereby primary productivity and plant community composition [19���21]. Moreover, soil biota comprise some of the most deleterious herbivores and pathogens often emerging as destruc- tive pests [20,22]. Although this knowledge provoked multiple studies on the relationship between aboveground (plant) and belowground diversity and functions, the significance of this relationship still is disputed [10,23���25]. The question arises how plant diversity affects soil biota and why there is this lack of consistency and mechanistic understand- ing. For instance, there is an ongoing debate on the relevance of plant species richness versus that of plant species identity or presence of key plant functional groups for soil biota and functions [11,23,25,26]. Since virtually all soil organisms are heterotrophs, and thus essentially rely on the quantity and quality of plant derived residues entering the soil subsystem [20,27,28], declining plant diversity, accompanied by deterioration of resource diversity, quantity and quality, is likely to impact the density, diversity and functioning of soil biota [10,25]. Hooper et al. [10] suggested a step-by-step hypothesis how the diversity of primary producers results in higher belowground diversity assuming strong bottom-up control of biodiversity in soil communities. In essence, increased diversity of plant derived resources increases the diversity of decomposers and herbivores in soil, which in turn promotes the diversity of other components of the soil food web. Moreover, an additional causal relationship between above- and belowground diversity may arise from enhanced microhabitat diversity in complex plant communities [29]. Acknowledging the mixed evidence on the correlation between above- and belowground density and diversity, Hooper et al. [10] highlighted the need to acquire a mechanistic understanding of this relationship and ascribed this topic top research priority. Most previous studies highlighted the significance of key plant functional groups for the performance of soil biota. Particularly legumes enhance the fertility of soils by N2 fixation and the input of high quality (nitrogen-rich) litter materials [24,26,30���32]. Positive impacts of plant diversity thus often have been ascribed to the increased probability of including legume species in more diverse plant communities [24,26,33], i.e. the selection or sampling effect of plant diversity [34,35]. Moreover, increased performance of soil biota has been attributed to elevated primary productivity [11,24]. Recent studies indicate that missing or inconsistent responses of soil biota to plant diversity may have been due to belowground legacy effects and the short-term character of most studies [25,26,36]. Indeed, most studies are based on snapshot measures and detailed investigations of soil biota in time are extremely scarce [26,37]. Further, only few studies considered a wide range of taxonomic or functional groups of soil biota in a single experiment [11,24,26,33,36] complicating the comparability of results. In addition, the observed variability in the response of soil biota to plant diversity may have been due to the investigation of differential trophic positions relative to the manipulated level. Recent reports suggest that the effect of diversity loss decreases with the trophic distance from the manipulated level [8,26]. In order to improve our mechanistic understanding of plant diversity effects on soil biota, we repeatedly sampled soil microorganisms and soil fauna in the framework of the Jena Experiment, a large grassland plant diversity experiment in Germany [18]. We intended to provide a comprehensive overview of plant community effects on multiple functional components of soil biota, ranging from short-term (two years after establishment) to long-term effects (after six years). Moreover, the Jena Experiment offers the unique possibility to independently explore the impacts of plant species richness (1���60 species), plant functional group richness (1���4 groups), and presence of key plant functional groups. The block design and the high replication of the Jena Experiment allow accounting for soil heterogeneity effects [18] and to delineate genuine diversity effects [10]. Determination of plant productivity measures above and below the ground further allows exploring the relevance of mere biomass effects. We hypothesized that (1) the role of plant diversity as driver of soil biota increases with time thereby exceeding that of the presence of key plant functional groups, and (2) changes in plant diversity effects depend on the functional affiliation of soil biota with decomposers responding slowest due to soil legacy effects. Materials and Methods Experimental setup The study was conducted in the framework of the Jena Experiment, a large field experiment investigating the role of biodiversity for element cycling and trophic interactions in grassland communities [18]. The study site is located on the floodplain of the Saale river at the northern edge of the city of Jena (Thuringia, Germany). Mean annual air temperature 3 km south of the field site is 9.3uC and annual precipitation is 587 mm (Fig. S1 [38]). The site had been used as an arable field for the last 40 years and the soil is an Eutric Fluvisol. The experiment was established in May 2002 and the studied system represents Central European mesophilic grassland traditionally used as hay meadow (Arrhenatherion community). A pool of 60 native plant species was used to establish a gradient of plant species (1, 2, 4, 8, 16 and 60) and plant functional group richness (1, 2, 3 and 4) in a total of 82 plots of 20620 m (Table S1 [18]). Using above- and belowground morphological traits, phenological traits and N2 fixation ability, plant species were aggregated into four plant functional groups: grasses (16 species), small herbs (12 species), tall herbs (20 species), and legumes (12 species) [18]. Experimental plots were mown twice a year (June and September), as is typical for hay meadows, and weeded twice a year (April and July) to maintain the target species composition. Plots were assembled into four blocks following a gradient in soil characteristics, each block containing an equal number of plots of plant species and plant functional group richness levels. Further information on the design and setup of the Jena Experiment is given in Roscher et al. [18]. Soil biota Soil samples for soil microbial measurements were taken from all plots in May 2004, 2006 and 2008. Briefly, at each sampling campaign, five soil samples were taken to a depth of 5 cm using a metal corer (diameter 5 cm), pooled and stored at 5uC. Before measurement, soil samples were homogenized, sieved (2 mm) to remove larger roots, animals and stones [39] and adjusted to a gravimetric soil water content of 25%. Microbial biomass C (Cmic) was measured using an O2-microcompensation apparatus [40]. The microbial respiratory response was measured at hourly intervals for 24 h at 22uC. Substrate-induced respiration was calculated from the respiratory response to D-glucose [39]. Glucose was added according to preliminary studies to saturate the catabolic enzymes of microorganisms (4 mg g21 dry weight solved in 400 ml deionized water). The mean of the lowest three readings within the first 10 h was taken as maximum initial respiratory response (MIRR ml O2 h21 g21 soil dry weight) and microbial biomass (mg C g21 soil dry weight) was calculated as 38 6 MIRR [41]. Soil meso- and macrofauna were collected from soil cores taken to a depth of 10 cm in autumn 2004 (October), 2006 (November) and 2008 (October). Soil cores were taken using a steel corer (5 cm Plant Diversity Effects on Soil Biota PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16055