Short- and long-term effects of biodiversity on soil nutrient concentrations in a semi-natural grassland: results from a 14-year experiment

Global biodiversity is declining at an alarming rate, which is likely to have important consequences on ecosystem functioning. Previous studies have shown that in the short term, higher plant biodiversity in grasslands is linked to lower soil nitrogen concentrations, particularly of nitrate, probably due to higher plant uptake. It is unknown, however, how this trend will develop in the long term. To establish long-term responses to experimental changes in biodiversity, long-term data in adequately high resolution is required to separate the long-term trend from seasonal variation in the data, and such data sets are still exceedingly rare. We present a data set of soil solution nitrogen and phosphorus concentrations collected every two weeks over 14 years after the establishment of an experimental grassland with varying levels of biodiversity. Analysis of this data allows us to determine a) whether the system has reached a new steady-state in soil nutrients after conversion from cropland soils to semi-natural grassland 15 years ago, and b) whether these steady-states are different for different levels of plant biodiversity. Furthermore, we expect to be able to detect c) the effects of extreme events (drought, flood) and d) temporal trends under different levels of plant biodiversity before the establishment of steady state. This will have important implications for our understanding of both the biodiversity-ecosystem functioning relationship and the nutrient dynamics of soils changing from previously fertilized systems to semi-natural grasslands. Our results might additionally have practical implications for the establishment and management of hay meadows

Stable hydrogen isotope ratios in crystal water of clay minerals

Hydrogen is the most abundant element in the Universe. But the utilization of the H isotopic composition (δH-2 value) of soil to elucidate biogeochemical processes or to serve as a palaeo climate proxy is still in its infancy. In our research, we will focus on the δH-2 value of nonexchangeable H in the clay fraction of soils. The δH-2 value of structural H in clay minerals – mainly from C-poor subsoils - has been studied since the 1970s. The δH-2 value of clay minerals mainly depends on (a) the average δH-2 value of ambient water at the site and time of formation, and on (b) the size of the equilibrium isotopic fractionation factor between water and clay mineral at the temperature of formation. In our research, we will focus on the δH-2 value of nonexchangeable H in the clay fraction of soils. Only nonexchangeable H in in structural water of minerals preserves its inherited δH-2 value and does not exchange with water at temperatures usually occurring in soil environments at the Earth’s surface. Nonexchangeable H is bound in crystal water, which integrates the δH-2 value of soil water over several millennia. This is in turn determined by palaeoclimatic variations of the precipitation’s δH-2 signal with distinguishable shifts e.g., from Pleistocene to Holocene. For a global data set, Ruppenthal (2014) reported a close correlation of bulk soil δH-2 values with those of the mean local precipitation and confirmed this for organic matter, while the clay fraction of soils was up to now not studied. We will adapt a steam equilibration method with water vapor of known H isotopic composition – formerly applied by Ruppenthal (2014) on SOM and bulk soil – to clay fractions and compare our results to the hitherto used heating treatments (200-250°C) under vacuum. We expect that the δH-2 signal of the clay fraction of Bt horizons will serve to differentiate soils developed in different climatic epochs (e.g., Holocene, last interstadial, last interglacial) by analyzing dated palaeo soil samples. To test the hypothesis that there is a similar global regression line of the δH-2 values in structural water of clay as up to now reported for bulk soils and soil organic matter, we will analyze the clay fraction in a global set of soil samples

Stable hydrogen isotope ratios in soil organic matter

Stable H isotope ratios are a promising indicator of OM transformation processes (Schimmelmann et al., 2006). δ2H values of bulk organic matter and of specific organic compounds can be used as ecological tracer and forensic tool if the proportion of H that readily exchanges with ambient moisture is accounted for (Wassenaar & Hobson, 1998). There are a few reports about the H isotope ratios in plant-soil systems illustrating that there is little knowledge of the controls of the isotopic composition of the non-exchangeable H fraction of bulk OM (Schimmelmann et al., 2006; Ruppenthal et al., 2015). The increasingly closer relationship between δ2H values of rainfall and of non-exchangeable H in OM (δ2Hn) in the order, plant – plant litter (above- and belowground) – soil along a climatic gradient (Ruppenthal et al., 2015) suggests that decomposition influences δ2Hn values in OM in a systematic way. However, there are knowledge gaps concerning the fractionation factors and the extent of incorporation of ambient water-H into the nonexchangeable fraction of H in OM during decomposition. Our research will focus on the mechanisms responsible for the strong correlation between δ2H values in rainfall and δ2Hn values of SOM. Therefore, our study aims to investigate (1) the incorporation of ambient water-H into the nonexchangeable H fraction in OM during decomposition by heterotrophic bacteria as model organisms and quantify apparent fractionation factors, (2) the extent of incorporation of ambient water into the nonexchangeable H fraction of OM by the soil microbial community under laboratory conditions, and (3) the extent to which H is incorporated into nonexchangeable OM pool from ambient water during decomposition of aboveground litter under field conditions. We will work with microcosms using two bacteria species and determine decomposition rates of litter. Steam equilibration (Ruppenthal et al., 2015) and TC/EA-IRMS are used as analytical tools. We expect that different decomposition rates because of differences in litter quality will be reflected by the extent of H incorporation from ambient water into the nonexchangeable H fraction of the products. Additionally, different litter types enriched in 2H will be buried in soil of forest stands. We hypothesize that the incorporation of 2H-depleted ambient water into 2H-enriched nonexchangeable H fraction of OM will depend on litter type, soil moisture/ temperature, and the heterotrophic activity during the experiment

15N tracing to elucidate links between biodiversity and nitrogen cycling in a grassland experiment

Nitrogen (N) cycling is a fundamental ecosystem function of high complexity because N undergoes many transformations in soil and vegetation. The effect of biodiversity loss on ecosystem functions in general, and on N cycling in particular, was studied in several manipulative field experiments. To generate a comprehensive view of the influence of species richness on all major N transformations, we conducted laboratory incubations, in which we added 15N-labeled ammonium and nitrate to soil samples of the “Jena Experiment”, a manipulative large scale, long-term biodiversity experiment in grassland. The experimental site is located in Jena, Germany. The design consists of 4 blocks and 82 plots with 1-60 species and 1-4 functional groups (grasses, legumes, small herbs, tall herbs). Approx. 400 g of field-fresh soil was sampled from each plot of one of the 4 blocks and divided into three aliquots of 100 g each. In order to trace N turnover, we amended the incubations (in triplicate) either with 15N-labelled (98 at%) ammonium, nitrate, or with a mixture of both. The samples were incubated for two months at 20°C. Soil solution was extracted 1, 2, 4, 9 and 16 days after 15N application by percolating 100 mL of nutrient solution through each vessel. Concentrations of NH4-N, NO3-N and total N in the extracts were determined with colorimetric methods. The N-isotopic composition in nitrate was analyzed by isotope ratio mass spectrometry (IRMS) using the denitrifier method. Ammonium N isotope ratios were determined using the “hypobromite oxidation” method, in which ammonium-N is converted to nitrite followed by azide reaction to nitrous oxide and IRMS analysis. The results will be comprehensively evaluated in a quantitative context using the modelling approach of Müller et al. (2007) to determine the size of six N pools and the rates of nine N transformations. Links between N transformation rates, N-pool size and plant species richness will be verified with the help of ANOVA

Relationships between ecosystem functions vary among years and plots and are driven by plant species richness

Ecosystem management aims at providing many ecosystem services simultaneously. Such ecosystem service multifunctionality can be limited by tradeoffs and increased by synergies among the underlying ecosystem functions (EF), which need to be understood to develop targeted management. Previous studies found differences in the correlation between EFs. We hypothesised that correlations between EFs are variable even under the controlled conditions of a field experiment and that seasonal and annual variation, plant species richness, and plot identity (identity effects of plots, such as the presence and proportion of functional groups) are drivers of these correlations. We used data on 31 EFs related to plants, consumers, and physical soil properties that were measured over 5 to 19 years, up to three times per year, in a temperate grassland experiment with 80 different plots, constituting six sown plant species richness levels (1, 2, 4, 8, 16, 60 species). We found that correlations between pairs of EFs were variable, and correlations between two particular EFs could range from weak to strong or negative to positive correlations among the repeated measurements. To determine the drivers of pairwise EF correlations, the covariance between EFs was partitioned into contributions from species richness, plot identity, and time (including years and seasons). We found that most of the covariance for synergies was explained by species richness (26.5%), whereas for tradeoffs, most covariance was explained by plot identity (29.5%). Additionally, some EF pairs were more affected by differences among years and seasons, showing a higher temporal variation. Therefore, correlations between two EFs from single measurements are insufficient to draw conclusions on tradeoffs and synergies. Consequently, pairs of EFs need to be measured repeatedly under different conditions to describe their relationships with more certainty and be able to derive recommendations for the management of grasslands

Biodiversity effects on ecosystem functioning in a 15-year grassland experiment: Patterns, mechanisms, and open questions

In the past two decades, a large number of studies have investigated the relationship between biodiversity and ecosystem functioning, most of which focussed on a limited set of ecosystem variables. The Jena Experiment was set up in 2002 to investigate the effects of plant diversity on element cycling and trophic interactions, using a multi-disciplinary approach. Here, we review the results of 15 years of research in the Jena Experiment, focussing on the effects of manipulating plant species richness and plant functional richness. With more than 85,000 measures taken from the plant diversity plots, the Jena Experiment has allowed answering fundamental questions important for functional biodiversity research. First, the question was how general the effect of plant species richness is, regarding the many different processes that take place in an ecosystem. About 45% of different types of ecosystem processes measured in the ‘main experiment’, where plant species richness ranged from 1 to 60 species, were significantly affected by plant species richness, providing strong support for the view that biodiversity is a significant driver of ecosystem functioning. Many measures were not saturating at the 60-species level, but increased linearly with the logarithm of species richness. There was, however, great variability in the strength of response among different processes. One striking pattern was that many processes, in particular belowground processes, took several years to respond to the manipulation of plant species richness, showing that biodiversity experiments have to be long-term, to distinguish trends from transitory patterns. In addition, the results from the Jena Experiment provide further evidence that diversity begets stability, for example stability against invasion of plant species, but unexpectedly some results also suggested the opposite, e.g. when plant communities experience severe perturbations or elevated resource availability. This highlights the need to revisit diversity–stability theory. Second, we explored whether individual plant species or individual plant functional groups, or biodiversity itself is more important for ecosystem functioning, in particular biomass production. We found strong effects of individual species and plant functional groups on biomass production, yet these effects mostly occurred in addition to, but not instead of, effects of plant species richness. Third, the Jena Experiment assessed the effect of diversity on multitrophic interactions. The diversity of most organisms responded positively to increases in plant species richness, and the effect was stronger for above- than for belowground organisms, and stronger for herbivores than for carnivores or detritivores. Thus, diversity begets diversity. In addition, the effect on organismic diversity was stronger than the effect on species abundances. Fourth, the Jena Experiment aimed to assess the effect of diversity on N, P and C cycling and the water balance of the plots, separating between element input into the ecosystem, element turnover, element stocks, and output from the ecosystem. While inputs were generally less affected by plant species richness, measures of element stocks, turnover and output were often positively affected by plant diversity, e.g. carbon storage strongly increased with increasing plant species richness. Variables of the N cycle responded less strongly to plant species richness than variables of the C cycle. Fifth, plant traits are often used to unravel mechanisms underlying the biodiversity–ecosystem functioning relationship. In the Jena Experiment, most investigated plant traits, both above- and belowground, were plastic and trait expression depended on plant diversity in a complex way, suggesting limitation to using database traits for linking plant traits to particular functions. Sixth, plant diversity effects on ecosystem processes are often caused by plant diversity effects on species interactions. Analyses in the Jena Experiment including structural equation modelling suggest complex interactions that changed with diversity, e.g. soil carbon storage and greenhouse gas emission were affected by changes in the composition and activity of the belowground microbial community. Manipulation experiments, in which particular organisms, e.g. belowground invertebrates, were excluded from plots in split-plot experiments, supported the important role of the biotic component for element and water fluxes. Seventh, the Jena Experiment aimed to put the results into the context of agricultural practices in managed grasslands. The effect of increasing plant species richness from 1 to 16 species on plant biomass was, in absolute terms, as strong as the effect of a more intensive grassland management, using fertiliser and increasing mowing frequency. Potential bioenergy production from high-diversity plots was similar to that of conventionally used energy crops. These results suggest that diverse ‘High Nature Value Grasslands’ are multifunctional and can deliver a range of ecosystem services including production-related services. A final task was to assess the importance of potential artefacts in biodiversity–ecosystem functioning relationships, caused by the weeding of the plant community to maintain plant species composition. While the effort (in hours) needed to weed a plot was often negatively related to plant species richness, species richness still affected the majority of ecosystem variables. Weeding also did not negatively affect monoculture performance; rather, monocultures deteriorated over time for a number of biological reasons, as shown in plant-soil feedback experiments. To summarize, the Jena Experiment has allowed for a comprehensive analysis of the functional role of biodiversity in an ecosystem. A main challenge for future biodiversity research is to increase our mechanistic understanding of why the magnitude of biodiversity effects differs among processes and contexts. It is likely that there will be no simple answer. For example, among the multitude of mechanisms suggested to underlie the positive plant species richness effect on biomass, some have received limited support in the Jena Experiment, such as vertical root niche partitioning. However, others could not be rejected in targeted analyses. Thus, from the current results in the Jena Experiment, it seems likely that the positive biodiversity effect results from several mechanisms acting simultaneously in more diverse communities, such as reduced pathogen attack, the presence of more plant growth promoting organisms, less seed limitation, and increased trait differences leading to complementarity in resource uptake. Distinguishing between different mechanisms requires careful testing of competing hypotheses. Biodiversity research has matured such that predictive approaches testing particular mechanisms are now possible

The Evolution of Ecological Diversity in Acidobacteria

Acidobacteria occur in a large variety of ecosystems worldwide and are particularly abundant and highly diverse in soils. In spite of their diversity, only few species have been characterized to date which makes Acidobacteria one of the most poorly understood phyla among the domain Bacteria. We used a culture-independent niche modeling approach to elucidate ecological adaptations and their evolution for 4,154 operational taxonomic units (OTUs) of Acidobacteria across 150 different, comprehensively characterized grassland soils in Germany. Using the relative abundances of their 16S rRNA gene transcripts, the responses of active OTUs along gradients of 41 environmental variables were modeled using hierarchical logistic regression (HOF), which allowed to determine values for optimum activity for each variable (niche optima). By linking 16S rRNA transcripts to the phylogeny of full 16S rRNA gene sequences, we could trace the evolution of the different ecological adaptations during the diversification of Acidobacteria. This approach revealed a pronounced ecological diversification even among acidobacterial sister clades. Although the evolution of habitat adaptation was mainly cladogenic, it was disrupted by recurrent events of convergent evolution that resulted in frequent habitat switching within individual clades. Our findings indicate that the high diversity of soil acidobacterial communities is largely sustained by differential habitat adaptation even at the level of closely related species. A comparison of niche optima of individual OTUs with the phenotypic properties of their cultivated representatives showed that our niche modeling approach (1) correctly predicts those physiological properties that have been determined for cultivated species of Acidobacteria but (2) also provides ample information on ecological adaptations that cannot be inferred from standard taxonomic descriptions of bacterial isolates. These novel information on specific adaptations of not-yet-cultivated Acidobacteria can therefore guide future cultivation trials and likely will increase their cultivation success