Feral cattle (Bos taurus) and horses (Equus ferus caballus) are commonly introduced to European rewilding areas to halt vegetation succession and to conserve light-demanding species. Yet, we still do not understand how the habitat preference of animals shapes vegetation structure at the landscape scale. Here, we used spatial preference modeling to understand drivers of space-use based on GPS-collared horses and cattle in a 120-ha rewilding area in Denmark. Using a time series of a satellite-based vegetation productivity index, we tested the ability of animal space-use to explain changes in vegetation, as well as the trend of its spatial variability at the reserve scale, as a measure of landscape-scale vegetation heterogeneity. We expected that animal space-use would be driven mainly by topography and vegetation characteristics and that highly used areas with open vegetation would remain open. We, indeed, found that vegetation density and landscape connectivity were good predictors of space-use preference for both cattle and horses. Additionally, both cattle and horses were strongly attracted to an artificial shelter located inside the reserve, warranting consideration of the use and placement of artificial infrastructure. Space-use diverged during periods of resource scarcity emphasizing the value of introducing a variety of herbivore functional types for optimizing structural ecosystem heterogeneity. As expected, we found that cattle and horses slow down vegetation succession in highly used areas, as shown by the negative correlation between changes in growing season productivity and intensively used areas dominated by short herbaceous and shrubby vegetation. We could also show that the highly used areas showed the largest reductions and the fastest recovery in vegetation greenness following the pan-European drought in 2018. A ~2/3 reduction in herbivore population size subsequent to the drought was followed by a general greening of the landscape, but with no clear relationship with space-use intensity. Our study supports that trophic rewilding with year-round grazing can limit vegetation densification at the landscape scale under near-natural conditions. This is pertinent in the face of accelerating succession toward increasingly dark and tree-dominated vegetation in temperate Europe's natural areas, and the associated biodiversity loss.

Understanding the pre-Homo sapiens baseline of the temperate forest biome is crucial for interpreting present-day biodiversity patterns, ecosystem functioning, and guiding restoration. In this review, we synthesize palaeoecological and ecological evidence to reassess vegetation structure across humid-temperate Europe during the Neogene (23-2.6 Myr) and Quaternary (2.6 Myr-1900 CE). We integrate data from multiple proxies - including pollen, macrofossils, isotopic composition, dental ecometrics, microcharcoal, and ancient environmental DNA - to evaluate whether Europe's temperate biome was dominated by closed-canopy forests, open grassland or by more heterogeneous woodland-grassland mosaics. Converging lines of evidence indicate that mosaic woodland vegetation - landscapes combining open, loosely wooded, and closed-canopy patches - prevailed throughout these periods, likely in large part maintained by abundant wild large herbivores. Following the Late-Pleistocene megafaunal collapse, tree cover expanded under reduced herbivore pressure. From the Mesolithic onward, humans increasingly shaped vegetation through burning and hunting. Since the Neolithic, our ancestors partially recreated lost disturbance dynamics through the use of domestic herbivores but also introduced novel drivers that produced ecosystems without historical analogues. These results challenge the traditional paradigm of closed-canopy forests being the dominant natural vegetation type in Europe, showing that homogenous closed-canopy forests are a recent phenomenon that only became the dominant "natural" vegetation after the decline of wild large herbivores and the loss of historical cultural management. Recognizing the woodland-grassland mosaic biome as the dominant natural baseline has major implications for conservation, rewilding, and biodiversity restoration strategies that reflect the ecological and evolutionary history of the temperate zone.

Groundwater-dependent Ecosystems (GDEs) are critical biodiversity hotspots and drought refugia that are driven by complex groundwater and ecological interactions. The detection of GDEs in temperate regions remains challenging due to weak phenological contrasts with surrounding vegetation. We developed a Positive-Unlabeled (PU) machine learning framework to detect temperate GDEs in Denmark using minimal input variables: two Normalized Difference Vegetation Index (NDVI) variables derived from Sentinel-2 imagery, and Height Above Nearest Drainage. The approach exploits two NDVI signals: the drought-resilience of GDEs during dry summers (dry-season analogue), and their delayed spring green-up caused by waterlogged soils.Our model achieved 79% detection of field-mapped GDEs within Danish NATURA 2000 areas while identifying 7 times more potential GDE area (122.500 ha) and finds further 222.000 ha of GDEs in river valleys outside the monitored NATURA 2000 areas. Plausibility analysis using a Water table depth (WTD) dataset, and tree species dataset showed good agreement between labeled and predicted GDEs. Sensitivity analysis across different model parameterizations revealed predicted GDE coverage ranging from 22% to 65% of the study area, with true positive rates of 45% to 93%.The framework addresses key limitations in GDE mapping by requiring only positive training labels and few input variables. The model performs comparably to dryland GDE detection methods while operating without explicit dry season dynamics. These results establish a baseline for temperate GDE detection across regions, supporting land- and water-resources management, and compliance with environmental directives requiring GDE identification.

Abstract Introduction Herbivore‐mediated ecosystem processes are fundamental to biodiversity, yet have been greatly diminished by megafauna extinctions and replacement of wild herbivores by managed livestock. With increasing restoration ambitions—including the United Nations' (UN) Decade on Ecosystem Restoration and the European Union (EU) Nature Restoration Law—there is a need to set meaningful restoration targets for large mammalian herbivores. Objectives We quantify natural baselines for herbivore biomass and body‐size structure and describe the roles of predation and migration. We also propose a dual framework of baseline‐aligned targets and partial restoration targets to support restoration planning. Methods At a workshop, we identified the biomass, size distribution, predation, and migration of large herbivores as key elements of a natural baseline. We synthesized paleoecological evidence, data from near‐intact ecosystems, and rewilding projects to provide empirical estimates of biomass and size structure. Results Based on paleoecological and near‐intact ecosystems, we tentatively set natural herbivore biomass at approximately 5000–30,000 kg/km 2 , with small herbivores (10–100 kg) contributing approximately 5–10% of total biomass and large and megaherbivores (≥500 kg) approximately 60%. Current European reserves average just 800 kg/km 2 —an order of magnitude lower—and are strongly skewed toward small and mesoherbivores such as deer and wild boar. Conclusions Given current constraints, these targets are unlikely to be fully met. Partial restoration, however, will improve ecosystem processes and likely biodiversity, but knowledge gaps remain, including how deviations from natural baselines affect biodiversity.

Anthropogenic impacts are reshaping plant biodiversity patterns, yet how community-composition shifts track environmental change at large spatial and temporal scales remains unclear. Here, we quantified trends in community-mean plant ecological indicator values (light, temperature, soil moisture, soil nitrogen, and soil reaction) across European vegetation between 1960 and 2020. We used spatiotemporal interpolation based on 644,524 plots and analyzed 18,345 time series encompassing diverse habitats. We found a clear shift in community composition over the past six decades with a steep increase in nitrogen-demanding species across all main habitat types, accompanied by a moderate increase in shade-tolerant species. Forest communities shifted toward species associated with higher soil pH, while wetland communities showed a decline in moisture-dependent species over time. Conversely, temperature indicator values were largely stable, except for recent thermophilization in alpine habitats. Our results indicate a widespread trend toward denser vegetation driven by eutrophication and changes in management practices.

Historical records reveal significant biodiversity loss, but over such long timespans that human minds gradually adjust to depauperated biotas. We used 19th and early 20th -century Danish records (100–180 years old) to address this blind spot and enhance our understanding of flora and fauna changes beyond mere richness gains or losses. By analyzing species’ traits, habitat affinities, and environmental indicators, we identified lost habitat types. Compared to the present, 19th-century Danish landscapes were more heterogeneous, featuring open, wooded pastures, now largely replaced by dense plantation forests. Historical data demonstrated that dark diversity is easily underestimated when based on contemporary data. Historical butterfly faunas were much more species-rich and had a stronger affinity for grazed ecosystems than current communities. Declining plant species of conservation interest were more light- and moisture-demanding and less nutrient-affiliated and competitive than persisting species. Declining butterfly species were linked to host plants with restricted national distribution. Declining bird species were typically carnivorous, migratory, and associated with open habitats, particularly grasslands, coastal areas, and freshwater wetlands. Our findings may guide the ambition level and direction of restoration efforts. The results suggest a restoration focus on open, heterogenous woodland, grassland and wetland ecosystems with low nutrient status and natural disturbance regimes, including populations of large herbivores near carrying capacity. This contrasts sharply with today’s timber-focused plantation forests and conservation area management governed by agricultural subsidies.

Nativeness is a concept central to biodiversity conservation and invasion biology, but there are several problems related to a classic binary nativeness definition. Dilemmas arise from the dynamic nature of species' distribution ranges on longer time scales, and difficulties arise in the application to smaller regions defined by arbitrary borders, and limited knowledge of species' past distributions, their natural migration potential, and time or mode of arrival in an area.To remedy these shortcomings, we propose a graduated definition of nativeness, where non-native species in a focal territory are categorized as either 'near-native', 'distant-native' or 'alien' based on geographical proximity to areas where they are native, their migration potential and climatic niche. As an example, we apply the graduated definition to the vascular flora of Denmark, reclassifying species as 'native', 'near-native' (native to countries in close vicinity, within their natural migration potential and with similar climatic niche to the focal territory), 'distant-native' (native to countries in the rest of Europe) or 'alien' (native to other continents). A change in nativeness status for single species could have practical management implications. More importantly, taking the non-equilibrium spatio-temporal nature of plant distributions into account allows for a more consistent concept of nativeness, which has the flexibility to accommodate future changes in species distributions induced by climate change.

The extensive, prehistoric loss of megafauna during the last 50 000 years led early naturalists to build the founding theories of ecology based on already-degraded ecosystems. In this article, we outline how large herbivores affect community ecology, with a special focus on plants, through changes to selection, speciation, drift, and dispersal, thereby directly impacting ecosystem diversity and functionality. However, attempts to quantify effects of large herbivores on ecosystem processes are markedly scarce in past and contemporary studies. We expect this is due to the shifting baseline syndrome, where ecologists omit the now-missing effects of extinct, large herbivores when designing experiments and theoretical models, despite evidence that large herbivores shaped the physical structure, biogeochemistry, and species richness of the studied systems. Here, we outline how effects of large herbivores can be incorporated into central theoretical models to integrate megaherbivore theory into community ecology. As anthropogenic impacts on climate and nutrient levels continue, further warping ecological processes and disconnecting species distributions from optimal conditions, the importance of quantifying large herbivore functionality, such as facilitation of dispersal and coexistence, increases. Our findings indicate that current scientific attention to large herbivores is disproportionate to their past impacts on habitat structure and evolutionary trajectories, as well as the role large herbivores can play in restoring diverse and resilient ecosystems.

Grazing by domestic herbivores is applied across Europe to combat the loss of light-dependent, species-rich communities due to encroachment by competitive woody and herbaceous plants. However, the billions of euros spent annually by the EU on grazing subsidies have failed to halt the loss of species in open habitats. We hypothesized that typical agri-environmental, seasonal grazing fails to simulate the ecological effects of now-extirpated, large, wild herbivores, which coevolved with these species-rich communities. We conducted a survey of 30 semi-natural sites, where grazing was either absent, seasonal, or year-round, across a spectrum of abiotic conditions. We recorded plant species diversity and cover and used plant traits to assess taxonomic and functional responses. Year-round grazing supported higher plant species richness and forb cover compared to seasonal or no grazing. Specifically, dormant-season (winter) grazing pressure increased species richness and forb cover, superseding additional effects of growing-season (summer) grazing pressure. Functional richness was similar across management types, likely due to higher plant trait similarity in year-round grazed sites. Our results support that dormant-season grazing plays a key role in weakening interspecific competition among plants and in enabling diverse forb communities to replace species-poor grass dominance. Synthesis and applications. Our results indicate that typical, seasonal grazing may be counterproductive in terms of promoting plant diversity. We found the most effective management strategy for conserving species-rich forb communities to be year-round grazing. Our results urge a greater focus on the ecological and evolutionary mechanisms behind the relationship between large herbivores and plants, not least the balance between grasses and forbs. We advocate a shift in conservation strategies towards natural grazing, to halt the continued loss of species that depend on open and semi-open, forb-rich habitats such as grasslands, wetlands, and woodlands.

Biodiversity change forecasts rely on long-term time series, but such data are often scarce in space and time. Here, we interpolated spatiotemporal changes in species richness using a new method based on machine learning that does not require temporal replication at sites. Using 698,692 one-time sampled vegetation plots, we estimated trends in vascular plant alpha diversity across Europe and validated our approach against 22,852 independent time series. We found an overall near-zero net change in species richness between 1960 and 2020. However, species richness generally declined from 1960 to 1980 and increased from 2000 to 2020 across habitats. Declines were most pronounced in forests, but trends varied across habitats and regions, with overall increases at higher latitudes and elevations, and declines or stable trends elsewhere. Our findings demonstrate how data without temporal replication can be used to reveal context-dependent biodiversity dynamics, underscoring their importance for conservation and management.

Liparis loeselii is a rare and declining orchid species restricted to rich fens in the Northern Hemisphere. Habitat destruction, eutrophication, drainage and scrub encroachment have been suggested as reasons for the decline. However, which factors are most important is not well understood. Based on vegetation and environmental properties from extant, potential and historical L. loeselii sites, we 1) developed habitat suitability models from either Ellenberg Indicator Values, which were derived from known habitat preferences of co-occurring plant species, or field-measured environmental properties, and 2) identified the primary reasons for the observed decline of L. loeselii. We found nutrient status to be the most important predictor for L. loeselii occurrence, followed by hydrology proxies (Ellenberg reaction and Ellenberg moisture). Vegetation height and Ellenberg light were of minor importance. Effect partitioning based on sites, from which L. loeselii has gone locally extinct, pinpointed eutrophication and drainage to be the most likely primary drivers of the species' demise. Phosphorus limitation induced by discharge of calcium-rich groundwater seems to be crucial for L. loeselii to sustain populations in landscapes dominated by intensive agriculture.

Aims: We introduce ReSurveyEurope - a new data source of resurveyed vegetation plots in Europe, compiled by a collaborative network of vegetation scientists. We describe the scope of this initiative, provide an overview of currently available data, governance, data contribution rules, and accessibility. In addition, we outline further steps, including potential research questions. Results: ReSurveyEurope includes resurveyed vegetation plots from all habitats. Version 1.0 of ReSurveyEurope contains 283,135 observations (i.e., individual surveys of each plot) from 79,190 plots sampled in 449 independent resurvey projects. Of these, 62,139 (78%) are permanent plots, that is, marked in situ, or located with GPS, which allow for high spatial accuracy in resurvey. The remaining 17,051 (22%) plots are from studies in which plots from the initial survey could not be exactly relocated. Four data sets, which together account for 28,470 (36%) plots, provide only presence/absence information on plant species, while the remaining 50,720 (64%) plots contain abundance information (e.g., percentage cover or cover-abundance classes such as variants of the Braun-Blanquet scale). The oldest plots were sampled in 1911 in the Swiss Alps, while most plots were sampled between 1950 and 2020. Conclusions: ReSurveyEurope is a new resource to address a wide range of research questions on fine-scale changes in European vegetation. The initiative is devoted to an inclusive and transparent governance and data usage approach, based on slightly adapted rules of the well-established European Vegetation Archive (EVA). ReSurvey:Europe data are ready for use, and proposals for analyses of the data set can be submitted at any time to the coordinators. Still, further data contributions are highly welcome.

Grazing at near-natural stocking rates is increasingly rare, whereas abandonment and overgrazing is common, despite both leading to loss of threatened species. Here, we evaluate a biodiversity-promotion strategy of a beef-producing company involving livestock grazing for conservation. Using field surveys, a national biodiversity map, and farmer interviews, we evaluate the conservation potential of farmers committing to a conservation grazing scheme. Most farmers practiced summer grazing, and-contrary to their pledge-primarily grazed areas of low conservation value, and with stocking rates far above carrying capacity. We conclude that a conservation grazing beef brand will only have real value for threatened species if committed to, and held accountable to, a scientifically informed grazing scheme. Our results point to rural traditions, legal regulations, and economic incentives as factors obstructing of a beneficial grazing practice. Hence, the results of this study call for a critical and scientifically informed approach to future land-sharing policies.

Aim: There is increasing interest in open- ended restoration with the focus on restor -ing natural processes rather than static compositional goals. Here, we investigated vegetation dynamics in response to three decades of trophic rewilding with large herbivores in a recent anthropogenic, fertile 55- km2 landscape on reclaimed marine sediments. This site provides important insights into plant community assembly in re-wilded, young ecosystems.Location: Oostvaardersplassen, the Netherlands.Methods: Using field surveys and vegetation data spanning two decades, we investi -gate vegetation dynamics of the mesic grasslands in Oostvaardersplassen, a pioneer trophic rewilding site in the Netherlands. To identify the effects of grazing and other ecological processes, we include comparisons to an adjacent ungrazed control site and to the dark diversity, that is, missing species expected to be present given the environ-ment and geography.Results: The impact of large herbivores has led to a more open and homogenous vegeta-tion with higher plant species richness in the mesic grasslands of Oostvaardersplassen compared to the ungrazed control. Compared to species in the dark diversity, the spe -cies present are more common in the region, more stress- tolerant, and less competi-tive, in line with joint effects of grazing and immigrational lag.Conclusions: The changes in vegetation composition and structure show that rewild-ing with large herbivores promotes the prevalence of stress- tolerant and low- staturedplant species, which are in decline in the broader landscape while reducing the domi-nance of otherwise widespread, competition-adapted species. The absence of numer -ous rare species suggests that dispersal limitation poses an important constraint for the diversity build- up in anthropogenically isolated natural areas, necessitating active species introductions to mitigate this issue in the short to medium term

QuestionHow does naturalistic grazing (trophic rewilding with large herbivores), in contrast to mowing and free succession (no grazing), affect plant community composition and species richness in temperate grassland grazed by semi-feral cattle and horses? LocationMols Laboratory, Denmark. MethodsWe investigated grazing exclosures in the rewilding area of the Mols Laboratory, four years after its establishment. We focused on moist to dry grassland vegetation, that is, excluding scrub and woodland. Each experimental block consisted of five 5 x 9 m plots, representing four fenced treatments, that is, summer-only grazing, winter-only grazing, full exclosure with annual autumn mowing and full exclosure with passive succession. The matrix (the fifth treatment) was grazed by large herbivores at close-to-natural densities, that is, regulated bottom-up by the carrying capacity of the area. Hence, even the seasonal grazing treatments were grazed at close-to-natural animal density. Quantitative plant community composition was assessed using the point-intercept method in 25 x 25 cm quadrats, supplemented with biomass calibration models based on additional quadrats, in which above-ground plant biomass was harvested after recording and the material sorted to species and weighed. Uniqueness was assessed as the sum of inverse range sizes for constituent species (unicity). ResultsWe found an appreciably higher plant species richness in grazing treatments than under both annual mowing and full exclosure, but only minor differences between seasonal grazing treatments. Uniqueness was highest in year-round and winter-only grazing and lowest in summer-only grazing. The forb:graminoid ratio tended to be high in the winter-only grazing treatment, whereas annual mowing was associated with dominance of graminoids over forbs. Full-exclosure plots had accumulation of litter and the lowest species richness. Initial heterogeneity between plots within blocks and a systematic difference between blocks in moist and dry grasslands may have diluted treatment effects at this early point after the onset of the experiment. Data analysis using the biomass estimates derived from the calibration models yielded only minor differences in the patterns described above, when compared to the results obtained using the raw number of intercepts. ConclusionsNaturalistic grazing is a goal in itself in ecological restoration, but also proposed as an efficient management tool to promote conservation of grassland plants and communities. We found both plant species richness and the prevalence of regionally rarer species (uni) to be higher with grazing than mowing or abandonment. Similarly, the tendency for forbs to prevail under grazing may translate into enhanced floral resources for anthophilous insects. Summer-only grazing at low density of large herbivores was not significantly different from winter-only and year-round grazing, but this treatment was much closer to natural grazing than intensive summer grazing typical of agri-environmental practices.

Abstract Biodiversity of soil is routinely assessed with environmental DNA—most often by massive parallel sequencing of marker genes (eDNA metabarcoding). Soil biodiversity may be investigated in relation to biodiversity research or as a tool in forensic investigations. After sampling, the taxonomic composition of soil biotic communities may change. In order to minimize community changes, it is desirable to reduce biological activity, e.g., by freezing immediately after sampling. However, this may be impossible due to remoteness of study sites or, in forensic cases, where soil has been attached to an item of interest for protracted periods of time. Here, we investigated the effect of storage duration and conditions on the assessment of the soil biota with eDNA metabarcoding. We extracted eDNA from freshly collected soil samples and again from the same samples after storage under contrasting temperature conditions and contrasting exposure (open/closed tubes). We used four different primer sets targeting bacteria, fungi, protists (cercozoans), and general eukaryotes. We quantified differences in richness, evenness, and community composition. Subsequently, we tested whether we could correctly infer habitat type and original sample identity after storage using a large reference dataset. We found stronger community composition differences with extended storage time and with higher storage temperature, and differences between open and closed tubes. However, for samples stored <28 days at a maximum of 20°C, changes were generally insignificant. Classification models successfully assigned most samples to their exact location of origin and correct habitat type even after 480 days storage. Even samples showing larger changes generally retained the original sample as the best match. For most biodiversity and forensic applications, storage of samples for days and even several weeks may thus not be a problem, if storage temperature does not exceed 20°C.

Brun et al. (2022) found that a few tall, high-SLA plant species had stronger effects on primary productivity than any measure of functional diversity.We add data on species rarity to show that ongoing biodiversity loss is unlikely to hamper ecosystem productivity, a core insight we feel the authors missed.

Fungal and arthropod consumers constitute the vast majority of global terrestrial biodiversity. Yet, the link from richness and composition of producer (plant) communities to the richness of consumer communities is poorly understood. Fungal and arthropod species richness could be a simple function of producer species richness at a site. Alternatively, it could be a complex function of chemical and structural properties of the producer species making up communities. We used databases on plant–fungus and plant–arthropod trophic links to derive the richness of consumer biota per associated plant species (coined link score). We assessed how well link scores could be predicted by simple attributes of plant species. Next, we used a multi‐taxon inventory of 130 sites, representing all major habitat types in a country (Denmark), to investigate whether link scores summed over plant species in communities (coined link sum) could outperform simple plant species richness as predictor of fungal and arthropod richness at the sites. We found plant species' link scores for both fungi and arthropods to be positively related to plant size, regional occupancy, nativeness and ectomycorrhizal status. Link‐based indices generally improved the prediction of richness of fungal and arthropod communities. For fungal communities, both observed link sum (from databases) and predicted link sum (from plant attributes) had high predictive power, while plant richness alone had none. For arthropod communities, predictive performance varied between functional groups. For both fungi and arthropods, richness predictions were further improved by considering abiotic habitat conditions. Our results underline the importance of plants as niche space for the megadiverse groups of arthropods and fungi. The plant–attribute approach holds promise for predicting local and regional consumer richness in areas of the world lacking detailed plant–consumer databases.

Study sites Our study sites were located in Kastbjerg Ådal (river valley) in Eastern Jutland, Denmark. It is within the Natura 2000 and habitat area no. 223 appointed because of the wide stretch of fens and mires among other qualities. The water course is in good ecological status according to the Water Framework Directive. Nitrogen deposition in this area is low to moderate, 12.5-14.5 kgN/ha/yr (Ellermann et al. 2021). Meadows and fens dominate the study area, known for ‘the longest stretch of rich fen’ in Denmark. Large parts of the river valley are heavily degraded by drainage, fertilization and scrub encroachment, but there have also been recent efforts to restore the watercourse and the valuable rich fens in the valley. Most fens and wet meadows have been abandoned and are now increasingly dominated by tall grasses, tall forbs and willow scrub, but summer grazing occurs in some areas and efforts are made to ensure grazing in the most valuable fens. The drier meadows are typically mown by heavy machinery. The sites were selected to represent gradients in soil moisture from moist to wet and gradients in nutrient status or productivity from poor to rich and included rich fens with characteristic species, fens dominated by Juncus subnodulosus and by Equisetum fluviatile, drained fens encroached by Phragmites australis and natural meadows with characteristic species and encroached by Epilobium hirsutum and meadows characterized by clovers and cultural grasses. The nine sites were of 10 m2, each with ten 1 m2 plots. The 10 plots within each site had treatments assigned randomly. Despite the location in the same river valley, the sites were considered independent because of their different management history and starting conditions and a typical inter-site distance of c. 225 meters. The experiment was established in June 2017 and treatments were repeated monthly during summer and bimonthly during winter, depending on treatment. Responses were recorded in July 2019. Experimental set-up and treatments Each of the 9 sites were divided into ten 1 m × 1 m plots each with a 0.5 m × 0.5 m inner square and a surrounding plot buffer zone with a control and the following treatments: burning, mowing, trampling, intensive summer grazing (SI), intensive summer grazing with trampling (SIT), extensive summer grazing (SE), extensive summer grazing with trampling (SET, year-round grazing (YR), and year-round grazing with trampling (YRT). Treatments were allocated randomly to each plot with the restriction that the control plot was always in one corner. The experiment was multifactorial with respect to grazing and trampling, whereas burning and mowing were stand-alone treatments. Initial biomass in each plot was estimated at the beginning of the experiment in June 2017 as follows: all standing biomass and litter was removed from the plots by manual cutting at the soil surface and following the micro-topography. Bryophytes were harvested by hand plucking. Biomass, litter and bryophytes from the plot buffer zone were cut separately from the inner square. To estimate the species abundances, a representative sample of the inner square was sorted into litter and live biomass (including bryophytes) by species as sorting the complete biomass was not feasible. All species, litter and biomass from the buffer zone were dried at 55° C and weighed. Using the relative abundance of species in the representative sample and with respect to the weight of the total biomass in the inner square, we estimated the abundance of the species in the inner square. Burning was simulated in March 2018 and 2019. We used wooden boards to shield and adjacent areas were watered before burning the focal plot with a gas weed burner. We burned on a calm day following a dry period with frost to ensure minimum risk of igniting underlying peat and fire spreading over ground, but ensuring that the standing biomass and litter would be dry enough to ignite. This is not a simulation of a naturally occurring wildfire, but corresponds to the conditions that managers would prefer for prescribed conservation burning at larger scales. We simulated mowing as a biomass removal in June 2018. Biomass was removed uniformly across the whole plot in a height of c. 5 cm depending on microtopography. This corresponds to conservation mowing in management but without the added disturbance and pressure from machines. Trampling disturbance was applied using short stilts that could be attached to the field biologist’s boot. The surface of the stilt was 49 cm2 which corresponds to a pressure of 1.3-1.5 kg/cm2 with the added weight of the field biologist. This again corresponds to the pressure of a hoof of cattle weighing c. 300-400 kg. Trampling was applied by stepping into the field randomly 60 times once every month from May to September and was the same treatment in combination with intensive, extensive and year round grazing. Grazing was simulated by cutting the above-ground biomass using a 1 m2 frame divided into a 10 cm coordinate system using the letters A-J on the x-axis and the numbers 1-10 on the y-axis. We cut tufts of biomass within the coordinate system using a list of random combinations of letters and numbers. This system enables “ungrazed” individuals to flower and set seeds. Based on our experience with grazing as an agri-environmental management practice in Denmark, we defined intensive summer grazing as taking place between May and September with the goal of removing all standing biomass by September. Extensive summer grazing also takes place May-September, but we carried this out at half the intensity as intensive summer grazing. Year-round grazing obviously takes place during the whole year (here administered May-September and November, January and March) with the goal of removing all standing biomass by the end of winter (March) before the beginning of a new growing season. We used the initial standing biomass (June 2017) as a measurement of plot productivity and estimated the amount of biomass to be removed during “grazing” as c. 20 % of the initial productivity each month May-September in intensive plots and with all standing biomass “grazed” in September. For extensive plots, we estimated removed biomass as c. 10 % of the initial productivity each month May-September leaving some standing biomass in September. Year-round grazing biomass removal was estimated as c. 10 % of yearly productivity removed every month May-September and November and 20 % removed in January and March resulting in no standing biomass at the end of the winter. As expected plot productivity changed as a result of the treatments, the amount of biomass removed had to be adjusted throughout the experiment. In practice, we aimed for removing twice the amount of biomass in intensive plots relative to extensive plots within the same site and always ensuring that no standing biomass was left in intensive plots in September, c. 50 % of the standing biomass was left in extensive plots in September and no standing biomass was left in year-round grazing plots in March (see actual removed biomass by treatment in Appendix A). All treatments were applied to the whole plot (1 m × 1 m), while the biomass response was only measured in the inner square (0.5 m × 0.5 m), leaving a buffer zone between plots with different treatments. Response variables A full plot (1 m × 1 m) species list was recorded in the field at the end of the experiment. From this total plot richness, vascular plant plot richness, bryophyte plot richness and number of indicator species per plot were calculated. Indicator species of conservation status are species considered moderately to very sensitive towards habitat degradation as defined by Fredshavn et al. (2010, see Appendix C). Indicator species are often adapted to relatively infertile habitats revealed by low Ellenberg N values and high Grime’s S values reflecting tolerance to nutrient shortage. Mean plot Grime’s C and S values (Grime et al. 1989) were calculated based on vascular plant species lists. We converted Grime’s life strategies to numerical values based on Ejrnæs and Bruun (2000). We performed a Nonmetric Multi-dimensional Scaling analysis (NMDS) on the presence-absence of vascular plant and bryophyte species at the end of the experiment using the function metaMDS in R-package ‘vegan’ (Oksanen et al. 2017) in R version 4.0.3 (R Core Team 2017), using Sørensen dissimilarity and a four-dimensional solution (k =4). The plot coordinates at the three first NMDS axes were extracted (NMS4 was discarded as noise) and these, along with the four richness variables as well as Grime’s C and S values, were used as response variables in Linear Mixed Models (LME) as described in ‘Statistical analyses’. Supplementary to regression models of single response variables we carried out a quadratic discriminant analysis (QDA) as described in ‘Statistical analyses’ using the change in six indicators during the course of the experiment. The difference between plot species richness at the beginning and end of the experiment was calculated based on the species lists from sorted initial biomass and end biomass (0.5 m × 0.5 m). Start-end differences were also calculated separately for vascular plant species richness, bryophyte species richness, richness of indicator species, the ratio between biomass of forbs and graminoids (grasses, sedges and rushes) and Grime’s C and S mean site values. Explanatory and co-variables Leaf nitrogen, carbon and phosphorous were determined from plot level sampling of leaf plates of grasses, i.e., the most abundant species group across sites. Fresh leaf plates were collected at the beginning and end of the project and then dried, ground and analyzed in the lab. Soil moisture (% volumetric water content) was measured as the mean of four measurements per plot at the beginning and end of the project using a FieldScout TDR 300 Soil Moisture Meter. The total number of species found in each site was used as a co-variable in species richness models reflecting the local species pool. Data processing All species names were checked for synonyms using the national database arter.dk. References: Ejrnæs, R. and H. H. Bruun (2000). "Gradient analysis of dry grassland vegetation in Denmark." Journal of Vegetation Science 11(4): 573-584. Ellermann, T., R. Bossi, J. Nygaard, J. H. Christensen, P. Løfstrøm, C. Monies, C. Geels, I. E. Nielsen and M. B. Poulsen (2021). Atmosfærisk deposition 2019. NOVANA. Aarhus, Aarhus Universitet, DCE - Nationalt Center for Miljø og Energi. Fredshavn, J., R. Ejrnæs and B. Nygaard (2010). "Teknisk anvisning for kortlægning af terrestriske naturtyper. TA-N3, Version 1.04. Fagdatacenter for Biodiversitet og Terrestriske Naturdata, Danmarks Miljøundersøgelser. 18 s. ." Grime, J. P., J. G. Hodgson and R. Hunt (1989). Comparative plant ecology: a functional approach to common British species. London, Unwin Hyman. Oksanen, J., F. G. Blanchet, R. Kindt, P. Legendre, R. B. O'Hara, G. L. Simpson, P. Solymos, M. H. H. Stevens and H. Wagner (2017). "Package 'vegan': Community Ecology Package. Version 2.4-3. http://cran.r-project.org/web/packages/vegan/vegan.pdf." R Core Team (2017). R: A language and environment for statistical computing. Vienna, Austria, R Foundation for Statistical Computing.