msGBS: A new high-throughput approach to quantify the relative species abundance in root samples of multispecies plant communities.

Plant interactions are as important belowground as aboveground. Belowground plant interactions are however inherently difficult to quantify, as roots of different species are difficult to disentangle. Although for a couple of decades molecular techniques have been successfully applied to quantify root abundance, root identification and quantification in multispecies plant communities remains particularly challenging. Here we present a novel methodology, multispecies genotyping by sequencing (msGBS), as a next step to tackle this challenge. First, a multispecies meta-reference database containing thousands of gDNA clusters per species is created from GBS derived High Throughput Sequencing (HTS) reads. Second, GBS derived HTS reads from multispecies root samples are mapped to this meta-reference which, after a filter procedure to increase the taxonomic resolution, allows the parallel quantification of multiple species. The msGBS signal of 111 mock-mixture root samples, with up to 8 plant species per sample, was used to calculate the within-species abundance. Optional subsequent calibration yielded the across-species abundance. The within- and across-species abundances highly correlated (R2 range 0.72-0.94 and 0.85-0.98, respectively) to the biomass-based species abundance. Compared to a qPCR based method which was previously used to analyse the same set of samples, msGBS provided similar results. Additional data on 11 congener species groups within 105 natural field root samples showed high taxonomic resolution of the method. msGBS is highly scalable in terms of sensitivity and species numbers within samples, which is a major advantage compared to the qPCR method and advances our tools to reveal hidden belowground interactions.

Effects of extreme rainfall events are independent of plant species richness in an experimental grassland community.

Global climate models predict more frequent periods of drought stress alternated by heavier, but fewer rainfall events in the future. Biodiversity studies have shown that such changed drought stress may be mitigated by plant species richness. Here, we investigate if grassland communities, differing in species richness, respond differently to climatic extremes within the growing season. In a 3-year outdoor mesocosm experiment, four grassland species in both monoculture and mixture were subjected to a rainfall distribution regime with two levels: periods of severe drought in the summer intermitted by extreme rainfall events versus regular rainfall over time. Both treatments received the same amount of water over the season. Extreme rainfall combined with drought periods resulted in a 15% decrease in aboveground biomass in the second and third year, compared to the regular rainfall regime. Root biomass was also reduced in the extreme rainfall treatment, particularly in the top soil layer (- 40%). All species developed higher water use efficiencies (less negative leaf δ13C) in extreme rainfall than in regular rainfall. These responses to the rainfall/drought treatment were independent of species richness, although the mixtures were on an average more productive in terms of biomass than the monocultures. Our experimental results suggest that mixtures are similarly able to buffer these within-season rainfall extremes than monocultures, which contrasts with findings in the studies on natural droughts. Our work demonstrates the importance of investigating the interactions between rainfall distribution and drought periods for understanding effects of climate change on plant community performance.

Lost in diversity: the interactions between soil-borne fungi, biodiversity and plant productivity.

There is consensus that plant species richness enhances plant productivity within natural grasslands, but the underlying drivers remain debated. Recently, differential accumulation of soil-borne fungal pathogens across the plant diversity gradient has been proposed as a cause of this pattern. However, the below-ground environment has generally been treated as a 'black box' in biodiversity experiments, leaving these fungi unidentified. Using next generation sequencing and pathogenicity assays, we analysed the community composition of root-associated fungi from a biodiversity experiment to examine if evidence exists for host specificity and negative density dependence in the interplay between soil-borne fungi, plant diversity and productivity. Plant species were colonised by distinct (pathogenic) fungal communities and isolated fungal species showed negative, species-specific effects on plant growth. Moreover, 57% of the pathogenic fungal operational taxonomic units (OTUs) recorded in plant monocultures were not detected in eight plant species plots, suggesting a loss of pathogenic OTUs with plant diversity. Our work provides strong evidence for host specificity and negative density-dependent effects of root-associated fungi on plant species in grasslands. Our work substantiates the hypothesis that fungal root pathogens are an important driver of biodiversity-ecosystem functioning relationships.

Spatial heterogeneity of plant-soil feedback affects root interactions and interspecific competition.

Plant-soil feedback is receiving increasing interest as a factor influencing plant competition and species coexistence in grasslands. However, we do not know how spatial distribution of plant-soil feedback affects plant below-ground interactions. We investigated the way in which spatial heterogeneity of soil biota affects competitive interactions in grassland plant species. We performed a pairwise competition experiment combined with heterogeneous distribution of soil biota using four grassland plant species and their soil biota. Patches were applied as quadrants of 'own' and 'foreign' soils from all plant species in all pairwise combinations. To evaluate interspecific root responses, species-specific root biomass was quantified using real-time PCR. All plant species suffered negative soil feedback, but strength was species-specific, reflected by a decrease in root growth in own compared with foreign soil. Reduction in root growth in own patches by the superior plant competitor provided opportunities for inferior competitors to increase root biomass in these patches. These patterns did not cascade into above-ground effects during our experiment. We show that root distributions can be determined by spatial heterogeneity of soil biota, affecting plant below-ground competitive interactions. Thus, spatial heterogeneity of soil biota may contribute to plant species coexistence in species-rich grasslands.

Early root overproduction not triggered by nutrients decisive for competitive success belowground.

BACKGROUND: Theory predicts that plant species win competition for a shared resource by more quickly preempting the resource in hotspots and by depleting resource levels to lower concentrations than its competitors. Competition in natural grasslands largely occurs belowground, but information regarding root interactions is limited, as molecular methods quantifying species abundance belowground have only recently become available. PRINCIPAL FINDINGS: In monoculture, the grass Festuca rubra had higher root densities and a faster rate of soil nitrate depletion than Plantago lanceolata, projecting the first as a better competitor for nutrients. However, Festuca lost in competition with Plantago. Plantago not only replaced the lower root mass of its competitor, but strongly overproduced roots: with only half of the plants in mixture than in monoculture, Plantago root densities in mixture were similar or higher than those in its monocultures. These responses occurred equally in a nutrient-rich and nutrient-poor soil layer, and commenced immediately at the start of the experiment when root densities were still low and soil nutrient concentrations high. CONCLUSIONS/SIGNIFICANCE: Our results suggest that species may achieve competitive superiority for nutrients by root growth stimulation prior to nutrient depletion, induced by the presence of a competitor species, rather than by a better ability to compete for nutrients per se. The root overproduction by which interspecific neighbors are suppressed independent of nutrient acquisition is consistent with predictions from game theory. Our results emphasize that root competition may be driven by other mechanisms than is currently assumed. The long-term consequences of these mechanisms for community dynamics are discussed.