Arthropod decline in grasslands and forests is associated with landscape-level drivers.

Recent reports of local extinctions of arthropod species1, and of massive declines in arthropod biomass2, point to land-use intensification as a major driver of decreasing biodiversity. However, to our knowledge, there are no multisite time series of arthropod occurrences across gradients of land-use intensity with which to confirm causal relationships. Moreover, it remains unclear which land-use types and arthropod groups are affected, and whether the observed declines in biomass and diversity are linked to one another. Here we analyse data from more than 1 million individual arthropods (about 2,700 species), from standardized inventories taken between 2008 and 2017 at 150 grassland and 140 forest sites in 3 regions of Germany. Overall gamma diversity in grasslands and forests decreased over time, indicating loss of species across sites and regions. In annually sampled grasslands, biomass, abundance and number of species declined by 67%, 78% and 34%, respectively. The decline was consistent across trophic levels and mainly affected rare species; its magnitude was independent of local land-use intensity. However, sites embedded in landscapes with a higher cover of agricultural land showed a stronger temporal decline. In 30 forest sites with annual inventories, biomass and species number-but not abundance-decreased by 41% and 36%, respectively. This was supported by analyses of all forest sites sampled in three-year intervals. The decline affected rare and abundant species, and trends differed across trophic levels. Our results show that there are widespread declines in arthropod biomass, abundance and the number of species across trophic levels. Arthropod declines in forests demonstrate that loss is not restricted to open habitats. Our results suggest that major drivers of arthropod decline act at larger spatial scales, and are (at least for grasslands) associated with agriculture at the landscape level. This implies that policies need to address the landscape scale to mitigate the negative effects of land-use practices.

Climate-land-use interactions shape tropical mountain biodiversity and ecosystem functions.

Agriculture and the exploitation of natural resources have transformed tropical mountain ecosystems across the world, and the consequences of these transformations for biodiversity and ecosystem functioning are largely unknown1-3. Conclusions that are derived from studies in non-mountainous areas are not suitable for predicting the effects of land-use changes on tropical mountains because the climatic environment rapidly changes with elevation, which may mitigate or amplify the effects of land use4,5. It is of key importance to understand how the interplay of climate and land use constrains biodiversity and ecosystem functions to determine the consequences of global change for mountain ecosystems. Here we show that the interacting effects of climate and land use reshape elevational trends in biodiversity and ecosystem functions on Africa's largest mountain, Mount Kilimanjaro (Tanzania). We find that increasing land-use intensity causes larger losses of plant and animal species richness in the arid lowlands than in humid submontane and montane zones. Increases in land-use intensity are associated with significant changes in the composition of plant, animal and microorganism communities; stronger modifications of plant and animal communities occur in arid and humid ecosystems, respectively. Temperature, precipitation and land use jointly modulate soil properties, nutrient turnover, greenhouse gas emissions, plant biomass and productivity, as well as animal interactions. Our data suggest that the response of ecosystem functions to land-use intensity depends strongly on climate; more-severe changes in ecosystem functioning occur in the arid lowlands and the cold montane zone. Interactions between climate and land use explained-on average-54% of the variation in species richness, species composition and ecosystem functions, whereas only 30% of variation was related to single drivers. Our study reveals that climate can modulate the effects of land use on biodiversity and ecosystem functioning, and points to a lowered resistance of ecosystems in climatically challenging environments to ongoing land-use changes in tropical mountainous regions.

Systematic Analysis of the Effect of Genomic Knock-Out of Non-Essential Promiscuous HAD-Like Phosphatases YcsE, YitU and YwtE on Flavin and Adenylate Content in Bacillus Subtilis.

Studying the metabolic role of non-essential promiscuous enzymes is a challenging task, as genetic manipulations usually do not reveal at which point(s) of the metabolic network the enzymatic activity of such protein is beneficial for the organism. Each of the HAD-like phosphatases YcsE, YitU and YwtE of Bacillus subtilis catalyzes the dephosphorylation of 5-amino-6-ribitylamino-uracil 5'-phosphate, which is essential in the biosynthesis of riboflavin. Using CRISPR technology, we have found that the deletion of these genes, individually or in all possible combinations failed to cause riboflavin auxotrophy and did not result in significant growth changes. Analysis of flavin and adenylate content in B. subtilis knockout mutants showed that (i) there must be one or several still unidentified phosphatases that can replace the deleted proteins; (ii) such replacements, however, cannot fully restore the intracellular content of any of three flavins studied (riboflavin, FMN, FAD); (iii) whereas bacterial fitness was not significantly compromised by mutations, the intracellular balance of flavins and adenylates did show some significant changes.

Shining a Spotlight on Methyl Groups: Photochemically Induced Dynamic Nuclear Polarization Spectroscopy of 5-Deazariboflavin and Its Nor Analogs.

5-Deazaflavins are analogs of naturally occurring flavin cofactors. They serve as substitutes for natural flavin cofactors to investigate and modify the reaction pathways of flavoproteins. Demethylated 5-deazaflavins are potential candidates for artificial cofactors, allowing us to fine-tune the reaction kinetics and absorption characteristics of flavoproteins. In this contribution, demethylated 5-deazariboflavin radicals are investigated (1) to assess the influence of the methyl groups on the electronic structure of the 5-deazaflavin radical and (2) to explore their photophysical properties with regard to their potential as artificial cofactors. We determined the proton hyperfine structure of demethylated 5-deazariboflavins using photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy, as well as density functional theory (DFT). To provide context, we compare our findings to a study of flavin mononucleotide (FMN) derivatives. We found a significant influence of the methylation pattern on the absorption properties, as well as on the proton hyperfine coupling ratios of the xylene moiety, which appears to be solvent-dependent. This effect is enhanced by the replacement of N5 by C5-H in 5-deazaflavin derivatives compared to their respective flavin counterparts.

Parental environmental effects are common and strong, but unpredictable, in Arabidopsis thaliana.

The phenotypes of plants can be influenced by the environmental conditions experienced by their parents. However, there is still much uncertainty about how common and how predictable such parental environmental effects really are. We carried out a comprehensive experimental test for parental effects, subjecting plants of multiple Arabidopsis thaliana genotypes to 24 different biotic or abiotic stresses, or combinations thereof, and comparing their offspring phenotypes in a common environment. The majority of environmental stresses caused significant parental effects, with -35% to +38% changes in offspring fitness. The expression of parental effects was strongly genotype-dependent, and multiple environmental stresses often acted nonadditively when combined. The direction and magnitude of parental effects were unrelated to the direct effects on the parents: Some environmental stresses did not affect the parents but caused substantial effects on offspring, while for others, the situation was reversed. Our study demonstrates that parental environmental effects are common and often strong in A. thaliana, but they are genotype-dependent, act nonadditively, and are difficult to predict. We should thus be cautious with generalizing from simple studies with single plant genotypes and/or only few individual environmental stresses. A thorough and general understanding of parental effects requires large multifactorial experiments.

The roles of brain lipids and polar metabolites in the hypoxia tolerance of deep-diving pinnipeds.

Lipids make up more than half of the human brain's dry weight, yet the composition and function of the brain lipidome is not well characterized. Lipids not only provide the structural basis of cell membranes, but also take part in a wide variety of biochemical processes. In neurodegenerative diseases, lipids can facilitate neuroprotection and serve as diagnostic biomarkers. The study of organisms adapted to extreme environments may prove particularly valuable in understanding mechanisms that protect against stressful conditions and prevent neurodegeneration. The brain of the hooded seal (Cystophora cristata) exhibits a remarkable tolerance to low tissue oxygen levels (hypoxia). While neurons of most terrestrial mammals suffer irreversible damage after only short periods of hypoxia, in vitro experiments show that neurons of the hooded seal display prolonged functional integrity even in severe hypoxia. How the brain lipidome contributes to the hypoxia tolerance of marine mammals has been poorly studied. We performed an untargeted lipidomics analysis, which revealed that lipid species are significantly modulated in marine mammals compared with non-diving mammals. Increased levels of sphingomyelin species may have important implications for efficient signal transduction in the seal brain. Substrate assays also revealed elevated normoxic tissue levels of glucose and lactate, which suggests an enhanced glycolytic capacity. Additionally, concentrations of the neurotransmitters glutamate and glutamine were decreased, which may indicate reduced excitatory synaptic signaling in marine mammals. Analysis of hypoxia-exposed brain tissue suggests that these represent constitutive mechanisms rather than an induced response towards hypoxic conditions.

Booster-microchannel plate (BMCP) detector for signal amplification in MALDI-TOF mass spectrometry for ions beyond m/z 50 000.

Top-down proteomics deals with the characterization of intact biomolecules, which reduces the sample complexity and facilitates the detection of modifications at the protein level. The combination of the matrix-assisted laser desorption/ionization (MALDI) technique with time-of-flight (TOF) mass analysis allows for the generation of gaseous ions in low charge states from high-mass biomolecules, followed by their mass-to-charge ratio (m/z) separation, as high-mass ions drift down the flight tube more slowly than lighter ones. However, the detection efficiency of conventional microchannel plate (MCP) detectors is strongly reduced with decreasing ion velocity-corresponding to an increase in ion mass-which impedes the reliable detection of high-mass biomolecules. Herein, we present a simple modification of the MCP detector that allows for the amplification of the signal from ionized proteins of up to m/z 150 000. Two circular electrodes were assembled in front of the conventional detector and set to negative electrical voltages to affect the positively charged ions directly before they impinge on the MCP, possibly through a combination of a velocity boost and ion optical effects. In the present study, three booster electrode configurations were experimentally tested to maximize the signal intensification. Compared to the conventional MCP assembly, the signal intensity was amplified in a proof-of-concept experiment by a factor of 24.3 and of 10.7 for the singly charged BSA ion (m/z 66 400) and for the singly charged IgG ion (m/z 150 000), respectively, by applying the booster-MCP (BMCP) detector.

Land-use intensity alters networks between biodiversity, ecosystem functions, and services.

Land-use intensification can increase provisioning ecosystem services, such as food and timber production, but it also drives changes in ecosystem functioning and biodiversity loss, which may ultimately compromise human wellbeing. To understand how changes in land-use intensity affect the relationships between biodiversity, ecosystem functions, and services, we built networks from correlations between the species richness of 16 trophic groups, 10 ecosystem functions, and 15 ecosystem services. We evaluated how the properties of these networks varied across land-use intensity gradients for 150 forests and 150 grasslands. Land-use intensity significantly affected network structure in both habitats. Changes in connectance were larger in forests, while changes in modularity and evenness were more evident in grasslands. Our results show that increasing land-use intensity leads to more homogeneous networks with less integration within modules in both habitats, driven by the belowground compartment in grasslands, while forest responses to land management were more complex. Land-use intensity strongly altered hub identity and module composition in both habitats, showing that the positive correlations of provisioning services with biodiversity and ecosystem functions found at low land-use intensity levels, decline at higher intensity levels. Our approach provides a comprehensive view of the relationships between multiple components of biodiversity, ecosystem functions, and ecosystem services and how they respond to land use. This can be used to identify overall changes in the ecosystem, to derive mechanistic hypotheses, and it can be readily applied to further global change drivers.

Detecting Pre-Analytically Delayed Blood Samples for Laboratory Diagnostics Using Raman Spectroscopy.

In this proof-of-principle study, we systematically studied the potential of Raman spectroscopy for detecting pre-analytical delays in blood serum samples. Spectra from 330 samples from a liver cirrhosis cohort were acquired over the course of eight days, stored one day at room temperature, and stored subsequently at 4 °C. The spectra were then used to train Convolutional Neural Networks (CNN) to predict the delay to sample examination. We achieved 90% accuracy for binary classification of the serum samples in the groups "without delay" versus "delayed". Spectra recorded on the first day could be distinguished clearly from all subsequent measurements. Distinguishing between spectra taken in the range from the second to the last day seems to be possible as well, but currently, with an accuracy of approximately 70% only. Importantly, filtering out the fluorescent background significantly reduces the precision of detection.

Artefact Profiling: Panomics Approaches for Understanding the Materiality of Written Artefacts.

This review explains the strategies behind genomics, proteomics, metabolomics, metallomics and isotopolomics approaches and their applicability to written artefacts. The respective sub-chapters give an insight into the analytical procedure and the conclusions drawn from such analyses. A distinction is made between information that can be obtained from the materials used in the respective manuscript and meta-information that cannot be obtained from the manuscript itself, but from residues of organisms such as bacteria or the authors and readers. In addition, various sampling techniques are discussed in particular, which pose a special challenge in manuscripts. The focus is on high-resolution, non-targeted strategies that can be used to extract the maximum amount of information about ancient objects. The combination of the various omics disciplines (panomics) especially offers potential added value in terms of the best possible interpretations of the data received. The information obtained can be used to understand the production of ancient artefacts, to gain impressions of former living conditions, to prove their authenticity, to assess whether there is a toxic hazard in handling the manuscripts, and to be able to determine appropriate measures for their conservation and restoration.

Expanding Reaction Horizons: Evidence of the 5-Deazaflavin Radical Through Photochemically Induced Dynamic Nuclear Polarization.

Deazaflavins are important analogues of the naturally occurring flavins: riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The use of 5-deazaflavin as a replacement coenzyme in a number of flavoproteins has proven particularly valuable in unraveling and manipulating their reaction mechanisms. It was frequently reported that one-electron-transfer reactions in flavoproteins are impeded with 5-deazaflavin as the cofactor. Based on these findings, it was concluded that the 5-deazaflavin radical is significantly less stable compared to the respective flavin semiquinone and quickly re-oxidizes or undergoes disproportionation. The long-standing paradigm of 5-deazaflavin being solely a two-electron/hydride acceptor/donor-"a nicotinamide in flavin clothing"-needs to be re-evaluated now with the indirect observation of a one-electron-reduced (paramagnetic) species using photochemically induced dynamic nuclear polarization (photo-CIDNP) 1 H nuclear magnetic resonance (NMR) under biologically relevant conditions.

Drug-Membrane Interactions: Effects of Virus-Specific RNA-Dependent RNA Polymerase Inhibitors Remdesivir and Favipiravir on the Structure of Lipid Bilayers.

The two RNA-dependent RNA polymerase inhibitors remdesivir and favipiravir were originally developed and approved as broad-spectrum antiviral drugs for the treatment of harmful viral infections such as Ebola and influenza. With the outbreak of the global SARS-CoV-2 pandemic, the two drugs were repurposed for the treatment of COVID-19 patients. Clinical studies suggested that the efficacy of the drugs is enhanced in the case of an early or even prophylactic application. Because the contact between drug molecules and the plasma membrane is essential for a successful permeation process of the substances and therefore for their intracellular efficiency, drug-induced effects on the membrane structure are likely and have already been shown for other substances. We investigated the impact of remdesivir and favipiravir on lipid bilayers in model and cell membranes via several biophysical approaches. The measurements revealed that the embedding of remdesivir molecules in the lipid bilayer results in a disturbance of the membrane structure of the tested phospholipid vesicles. Nevertheless, in a cell-based assay, the presence of remdesivir induced only weak hemolysis of the treated erythrocytes. In contrast, no experimental indication for an effect on the structure and integrity of the membrane was detected in the case of favipiravir. Regarding potential prophylactic or accompanying use of the drugs in the therapy of COVID-19, the physiologically relevant impacts associated with the drug-induced structural modifications of the membrane might be important to understand side effects and/or low effectivities.

Food metabolomics: Latest hardware-developments for nontargeted food authenticity and food safety testing.

The analytical requirements for food testing have increased significantly in recent years. On the one hand, because food fraud is becoming an ever-greater challenge worldwide, and on the other hand because food safety is often difficult to monitor due to the far-reaching trade chains. In addition, the expectations of consumers on the quality of food have increased, and they are demanding extensive information. Cutting-edge analytical methods are required to meet these demands. In this context, non-targeted metabolomics strategies using mass and nuclear magnetic resonance spectrometers (mass spectrometry [MS]) have proven to be very suitable. MS-based approaches are of particular importance as they provide a comparatively high analytical coverage of the metabolome. Accordingly, the efficiency to address even challenging issues is high. A variety of hardware developments, which are explained in this review, have contributed to these advances. In addition, the potential of future developments is highlighted, some of which are currently not yet commercially available or only used to a comparatively small extent but are expected to gain in importance in the coming years.

How 1,n-Bis(3-alkylimidazolium-1-yl) Alkane Interacts with the Phospholipid Membrane and Impacts the Toxicity of Dicationic Ionic Liquids.

Ionic liquids based on doubly charged cations, often termed dicationic ionic liquids (DILs), offer robust physicochemical properties and low toxicity than conventional monocationic ionic liquids. In this design-based study, we used solid-state NMR spectroscopy to provide the interaction mechanism of two DILs, 1,n-bis(3-alkylimidazolium-1-yl) alkane dibromide ([C2n(C7-nIM)2]2+·2Br-, n = 1, 6), with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) phospholipid membranes, to explain the low toxicity of DILs toward HeLa, Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae cell lines. Dications with a short linker and long terminal chains cause substantial perturbation to the bilayer structure, making them more membrane permeabilizing, as shown by fluorescence-based dye leakage assays. The structural perturbation is even higher than [C12(MIM)]+ monocations, which carry a single 12-carbon long chain and exhibit a much higher membrane affinity, permeability, and cytotoxicity. These structural details are a crucial contribution to the design strategies aimed at harnessing the biological activity of ionic liquids.

Land-use intensification causes multitrophic homogenization of grassland communities.

Land-use intensification is a major driver of biodiversity loss. Alongside reductions in local species diversity, biotic homogenization at larger spatial scales is of great concern for conservation. Biotic homogenization means a decrease in ß-diversity (the compositional dissimilarity between sites). Most studies have investigated losses in local (α)-diversity and neglected biodiversity loss at larger spatial scales. Studies addressing ß-diversity have focused on single or a few organism groups (for example, ref. 4), and it is thus unknown whether land-use intensification homogenizes communities at different trophic levels, above- and belowground. Here we show that even moderate increases in local land-use intensity (LUI) cause biotic homogenization across microbial, plant and animal groups, both above- and belowground, and that this is largely independent of changes in α-diversity. We analysed a unique grassland biodiversity dataset, with abundances of more than 4,000 species belonging to 12 trophic groups. LUI, and, in particular, high mowing intensity, had consistent effects on ß-diversity across groups, causing a homogenization of soil microbial, fungal pathogen, plant and arthropod communities. These effects were nonlinear and the strongest declines in ß-diversity occurred in the transition from extensively managed to intermediate intensity grassland. LUI tended to reduce local α-diversity in aboveground groups, whereas the α-diversity increased in belowground groups. Correlations between the ß-diversity of different groups, particularly between plants and their consumers, became weaker at high LUI. This suggests a loss of specialist species and is further evidence for biotic homogenization. The consistently negative effects of LUI on landscape-scale biodiversity underscore the high value of extensively managed grasslands for conserving multitrophic biodiversity and ecosystem service provision. Indeed, biotic homogenization rather than local diversity loss could prove to be the most substantial consequence of land-use intensification.

Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media.

Increasing demands for protein-based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed-batch processes. N-lactoyl-leucine and N-lactoyl-isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N-lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.

Recent hybrid speciation at the origin of the narrow endemic Pulmonaria helvetica.

BACKGROUND AND AIMS: Hybridization is known to drive plant speciation through the establishment of homoploid or allopolyploid hybrid species. Here we investigate the origin of Pulmonaria helvetica, a narrow endemic species described across a restricted area of Switzerland that was entirely covered by ice during the last glacial maximum. This species presents an original number of chromosomes (2n = 24) and morphological traits suggestive of a hybrid origin. METHODS: We sequenced a plastid locus and 1077 double-digest restriction-site-associated DNA (ddRAD) loci in 67 individuals from across the distribution range of P. helvetica and candidate progenitor species growing in the same area. Assignment of genotypes to main genetic clusters within and among taxa using STRUCTURE tested whether P. helvetica represents a genetically differentiated lineage and addressed the hypothesis of its hybrid origin. Comparative ecological modelling further addressed possible niche differentiation among taxa. KEY RESULTS: Pulmonaria helvetica was highlighted as a genetically homogeneous species distinct from co-occurring taxa. Consistent with a scenario of hybrid speciation, it presented clear evidence of balanced admixture between Pulmonaria officinalis (2n = 16) and Pulmonaria mollis s.l. (2n = 18, 22), which was also highlighted as a maternal progenitor based on plastid sequences. Limited genetic structure within the maternal progenitor is consistent with an origin of P. helvetica through either homoploid hybridization with considerable karyotype changes or via complex scenarios of allopolyploidy involving a dysploid taxon of P. mollis s.l. Comparative niche modelling indicated non-significant ecological differences between P. helvetica and its progenitors, supporting intrinsic factors resulting from hybridization as main drivers of speciation. CONCLUSIONS: Hybridization appears as a major process having promoted the postglacial origin of the narrow endemic P. helvetica, suggesting hybrid speciation as an effective process that rapidly produces new species under climate changes.

A DNA microarray for the authentication of giant tiger prawn (Penaeus monodon) and whiteleg shrimp (Penaeus (Litopenaeus) vannamei): a proof-of-principle.

This proof-of-principle study describes the development of a rapid and easy-to-use DNA microarray assay for the authentication of giant tiger prawns and whiteleg shrimp. Following DNA extraction and conventional end-point PCR of a 16S rDNA segment, the PCR products are hybridised to species-specific oligonucleotide probes on DNA microarrays located at the bottom of centrifuge tubes (ArrayTubes) and the resulting signal patterns are compared to those of reference specimens. A total of 21 species-specific probes were designed and signal patterns were recorded for 47 crustacean specimens belonging to 16 species of seven families. A hierarchical clustering of the signal patterns demonstrated the specificity of the DNA microarray for the two target species. The DNA microarray can easily be expanded to other important crustaceans. As the complete assay can be performed within half a day and does not require taxonomic expertise, it represents a rapid and simple alternative to tedious DNA barcoding and could be used by crustacean trading companies as well as food control authorities for authentication of crustacean commodities.

High-Throughput Selection and Characterisation of Aptamers on Optical Next-Generation Sequencers.

Aptamers feature a number of advantages, compared to antibodies. However, their application has been limited so far, mainly because of the complex selection process. 'High-throughput sequencing fluorescent ligand interaction profiling' (HiTS-FLIP) significantly increases the selection efficiency and is consequently a very powerful and versatile technology for the selection of high-performance aptamers. It is the first experiment to allow the direct and quantitative measurement of the affinity and specificity of millions of aptamers simultaneously by harnessing the potential of optical next-generation sequencing platforms to perform fluorescence-based binding assays on the clusters displayed on the flow cells and determining their sequence and position in regular high-throughput sequencing. Many variants of the experiment have been developed that allow automation and in situ conversion of DNA clusters into base-modified DNA, RNA, peptides, and even proteins. In addition, the information from mutational assays, performed with HiTS-FLIP, provides deep insights into the relationship between the sequence, structure, and function of aptamers. This enables a detailed understanding of the sequence-specific rules that determine affinity, and thus, supports the evolution of aptamers. Current variants of the HiTS-FLIP experiment and its application in the field of aptamer selection, characterisation, and optimisation are presented in this review.

Effects of fertilization and irrigation on vascular plant species richness, functional composition and yield in mountain grasslands.

Land-use intensification is a major threat to biodiversity in agricultural grasslands and fertilization is one of the main drivers. The effects of fertilization on biodiversity and plant functional composition (community-weighted mean traits and mean ecological indicator values) are well studied in lowland regions, but have received less attention in mountain grasslands. Moreover, in inner-alpine dry valleys, fertilizer is often applied in combination with irrigation, and irrigation effects are less well known. We experimentally tested the effects of fertilization and irrigation on vascular plant species richness and the functional composition of mountain grasslands in the Swiss Alps. After five years, fertilization increased yield but the relationship was quadratic with maximum yield reached at intermediate fertilizer levels (58 kg N ha-1year-1). The species richness of all vascular plants and forbs decreased, on average, by 6 and 5 species respectively, per 50 kg N of extra fertilizer (ha-1 year-1) applied. Fertilization also favored fast-growing plants (increased mean specific leaf area) and plants typically found in productive environments (increased mean indicator values for soil productivity and moisture). In contrast, we found no effects of irrigation on plant community composition, which suggests that irrigation does not affect vascular plant diversity to the same extent as fertilization in these mesic mountain hay meadows, at least in the mid-term. Our finding that maximum yield can be achieved at intermediate fertilizer levels is very important from an applied, agronomical and conservation point of view. It suggests that without loss of yield, farming costs and at the same time environmental pollution and negative effects on biodiversity can be reduced by applying less fertilizer. We therefore recommend maintaining non-intensive land use and keeping fertilizer inputs as low as possible to maintain the high plant diversity of mountain grasslands.