Restorative seeding controls annual invasive species, but perennials can thrive in the long term despite treatments in sand grassland restoration

Restorative seeding controls annual invasive species, but perennials can thrive in the long term despite treatments in sand grassland restoration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Restorative seeding controls annual invasive species, but perennials can thrive in the long term despite treatments in sand grassland restoration Nora Saradi, Bruna Paolinelli Reis, Edina Csákvári, Anna Cseperke Csonka, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4435901/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jun, 2025 Read the published version in Biological Invasions → Version 1 posted 5 You are reading this latest preprint version Abstract Biodiversity loss caused by invasive alien species is a major problem in planetary perspective. Ecological restoration is an important tool to counteract invasions, but invasive species may negatively affect restoration if present in the landscape. We investigated long-term changes in annual and perennial invasive alien species abundance in three sandy grassland restoration experiments. We evaluated the dependence of annual and perennial invasive species abundance on initial restoration intervention, invasive species propagule pressure from the surrounding landscape, and time since interventions. Restoration interventions (seeding, mowing and carbon amendment) were conducted at a total of eight sites in the Kiskunság region of Hungary. The interventions took place between 1995 and 2003 and were monitored for 17–25 years. To assess invasive propagule pressure around the experimental sites, total shoot numbers in adjacent 1 m by 1 m plots along 100-meter-long transects were counted in 2020–2021 from the center of the eight experimental sites. Invasive propagule pressure within a 100-meter buffer did not explain changes in the abundance of annual and perennial invasive species. The cover of annual invasive species has mostly decreased over time, and treatment (mainly seeding) could accelerate this process. The cover of perennial invasive species increased over time irrespective of applied treatments and landscape invasive propagule pressure. Our research showed that seeding with native species is an effective tool for restoring sandy grasslands and preventing the spread of annual invasive species, but our toolbox for preventing perennial invasion in grassland restoration is limited. dry grassland restoration invasive alien plant species invasive propagule pressure landscape impact long-term monitoring Figures Figure 1 Figure 2 Figure 3 Introduction One of the main causes of biodiversity loss is the presence and spread of invasive alien species (IPBES 2023). In most cases, the removal of invasive alien species is not followed by the recovery of degraded native habitats, therefore ecological restoration is considered increasingly vital in the fight against invasion (Pearson et al. 2016 ). Ecological restoration aims to help restore degraded, damaged or destroyed ecosystems and has proven to be suitable for improving the ecological integrity of degraded habitats (Martin 2017 ). In some instances, revegetation efforts can successfully suppress invasive plants (Halassy et al. 2023 ). However, restorative interventions can also lead to disturbances. Soil disturbance, burning, grazing, mowing and weed control are common restoration interventions which create open spaces in a community and increase the resources available to native species (Hobbs et al. 2007 , Flory and Clay 2009 , Papanastasis 2009 ). However, these types of disturbances can also mediate invasions (Hobbs and Huenneke 1992 ), especially when the invaders are disturbance-adapted species with good dispersal capacities (Funk et al. 2020 ). Therefore, in search of best restoration practices, we need to understand how invasive species respond to restorative treatments on the long term and how the presence of invasive species in the surrounding landscape influences the progress of restoration. Some studies have shown that different plant life forms respond differently to restorative interventions, which should therefore also be taken into consideration in management and restoration (Nyamai et al. 2011 , Halassy et al. 2021 ). Annual invasive species have a special set of traits that help them to successfully colonize new areas: they are characterized by efficient reproduction and dispersal, i.e. a wide variety of mixed dispersal vectors, and an appropriate germination strategy (e.g. biotic conditions are sensed by the seeds) can ensure their quick establishment in a new habitat (Fenesi & Botta-Dukát 2010 ). In contrast, for perennial invasive species, population expansion and adaptive evolution to new habitats can be much slower and more difficult, but longer residence times allow perennial invasive species to spread to more places, increasing the scale of invasion (Ni et al. 2021 ). Consequently, the two life form groups might require different approaches for nature conservation and restoration of the native habitat. For example, Ramula and colleagues ( 2008 ) found that manipulation of growth and reproductive mechanisms in annual invasive plants always resulted in population declines. However, in the case of perennial invasive species, neither influencing survival, growth nor reproductive mechanism separately was sufficient to reduce the population, while influencing survival and growth or survival and reproduction mechanisms together were more effective. Various forms of active restoration methods exist to mitigate the impact of invasive species and improve biodiversity (Weidlich et al. 2020 ). Mowing, one of the widely used restoration interventions has been shown to be efficient in reducing the density of perennial invasive plants even in the short term (Nagy et al. 2022 ), and it also strongly hinders the reproductive success of annual invasive species (Milakovic et al. 2014 ). Nevertheless, mowing and grazing can open up windows for colonization, which invasive species with good dispersal capacity can exploit first (Reis et al. 2021 ). Seeding has also been shown to be effective in reducing annual invasive species (Urza et al. 2019 ) and affecting the biomass production of perennial invasive species (Reinhardt and Galatowitsch 2008 ). However, indirect methods, such as soil nitrogen immobilization using carbon sources, have been less successful in controlling invasive species (Perry et al. 2010 ) and have been reported to affect mainly annual invasive species (Davis et al. 2000 ). Landscape factors can further modify the impact of restorative measures, therefore assessing and understanding landscape-scale impacts is essential for planning successful restoration interventions (Helsen et al. 2013 , Prach et al. 2015 ). Several studies have shown that the most important landscape features that influence the success of a restoration intervention are the area and perimeter of semi-natural habitat patches in the landscape and the proximity of propagule sources (Helsen et al. 2013 , Guido et al. 2016). This combined with high propagule pressure is one of the main factors influencing invasion (Catford et al. 2011 , Kröel-Dulay et al. 2019 ). There are habitats rich in native species that are optimal sources of natural recovery. However, in an anthropogenised landscape, many habitats are the sources of invasive species that can undermine restoration efforts (Holl and Aide 2011 , Vilà and Ibáñez 2011 , Csecserits et al. 2016 , Guido et al. 2016). Grasslands are one of the most vulnerable habitats as they are subject to the highest levels of plant invasion (Catford and Jones 2019 ). The most abundant invasive alien species are terrestrial plants, which represent nearly half of all invasive species in Europe (6 368 species) (Costello et al. 2022 ). Land-use change in general fosters plant invasions (Vilà and Ibáñez 2011 ), and grasslands were found to become more prone to invasion when traditional human interventions (e.g. grazing or mowing) are abandoned (Axmanová et al. 2021 ). According to a countrywide survey (called MÉTA), 5.5% of the territory of Hungary is covered by invasive perennial plants (not counting woody invaders) (Botta-Dukát 2008 ). The rate of land abandonment has accelerated in Hungary over the past three decades (Valkó et al. 2016 ), creating opportunities for the regeneration of abandoned lands, but also for the advance of invasive species (Török et al. 2003 ). Not surprisingly, grassland restoration has been ranked among the top 50 conservation research priorities (Mihók et al. 2015 , 2017 ) and the control of invasive species is in the focus of restorative interventions (Török et al. 2019 ). Methods for the restoration of Pannonian sandy grasslands are studied for about 20 years (Kiskun Restoration Experiments, KISKUN LTER). We applied mowing, carbon amendment and seeding after the cessation of arable cultivation or the clear-cutting of non-native plantations and monitored the outcome of restorative interventions in the long term (Halassy et al. 2016 ). Results so far show that the outcome of restoration interventions depends not only on the restorative intervention itself, but also on the elapsed time and the surrounding landscape (Reis et al 2022 ). Although a landscape impact is visible, there is a lack of knowledge about the distances from which the pressure of invasive alien species may pose a threat to the restoration efforts and on the different behavior of annual and perennial invasive species. Building on our previous research (Reis et al. 2022 ), in which we described the impact of landscape habitat composition on the long-term success of restoration, we used a finer scale transect method to study the impact of invasive propagule pressure from the neighborhoods surrounding the restoration sites on invasive species abundance and separately analyzed the response of annual and perennial invasive species at the restoration sites. The questions of our research are: (1) How does the level of annual and perennial invasion change with time in three sand grassland restoration experiments on the long term? (2) To what extent does the degree of invasion within the restoration experiments depend on the type of restoration intervention, the time since the intervention and the invasive propagule pressure from the landscape? Materials and methods Study area We collected long-term datasets from three different experiments, which were carried out at the Kiskun LTER site (Kiskun LTER 2005–2024), Hungary. The experiments are near three settlements (Bugac: 46°39'N, 19°36’E; Fülöpháza: 46°52'N, 19°24'E; Izsák: 46°45'N, 19°19'E) in the Kiskunság Sand Ridge (Fig. 1 ). The area lies in a harsh environment regarding the temperature, moisture, wind and soil conditions (Borhidi 1993 , Kovács-Láng et al. 2008 ). The average annual temperature is 10.5°C with large daily and annual fluctuations. The yearly precipitation is 520–540 mm, and drought events have become more frequent (especially during the summer). The predominant wind direction is NW to SE (Bagi 1990 ). The soils are Calcaric Arenosol with more than 90% of sand and less than 1% humus content (Zólyomi et al. 1997 , Kovács-Láng et al. 2000 , Buzási et al. 2021 ). The area is situated in the forest-steppe zone (Erdős et al. 2022 ) with a mosaic of different types of communities depending on topography and the parent material. One of its most typical components at the driest locations is the open sandy grassland ( Festucetum vaginatae ‘danubiale’ community ) . The Festucetum vaginatae ‘ danubiale’ is a semi-desert-like community, rich in endemic species and dominated by the tussock grasses Festuca vaginata Waldst. & Kit. ex Willd. and Stipa borysthenica Klokov ex Prokudin, which is present on the tops and Southern slopes of nutrient-poor and coarse-textured calcareous sand dunes (Kovács-Láng et al. 2000 ). Other typical natural habitat types are closed sand steppe, poplar-juniper shrubland, open and closed natural forest, and wetlands (mesotrophic wet meadows, Molinia -dominated meadows, and marshes) (Biró et al. 2013a ). Due to historical human land transformation, most of the present landscape is covered by agricultural areas (57%), forests - mostly non-native plantations − (19%), and settlements (6%) (CORINE 2000). Agricultural cultivation reached its peak during the socialist regime, in the 1960s. Afforestation by black and scots pine ( Pinus nigra J.F.Arnold, Pinus sylvestris L.), black locust ( Robinia pseudoacacia L. ) and native white poplar ( Popolus alba L.) was carried out during the 19—20th centuries to hold the sand. Land-use changes have led to the preservation of only approximately 19% of semi-natural habitats in the region. This includes not only remnants of primary habitats but also disturbed meadows and dry grasslands, some of which have been encroached by non-native trees (Biró et al. 2013a ). During the post-socialist transformation (1987–1999), the abandonment of agricultural land reached a significant level, especially in low productive areas, such as the Kiskunság, and besides the spontaneous recovery of native vegetation, invasive alien species began to spread to these abandoned areas (Biró et al. 2013a ; b ). Restoration experiments We conducted several experiments in the region with the aim to assist the recovery of sandy grassland habitats in degraded areas, and here we analyzed the invasion of non-native annual and perennial plant species in three of them offering long-term data. In the first experiment (EXP1), afforestation with non-native Robinia pseudoacacia caused grassland degradation. In order to restore grasslands, the trees were clear-cut in the winter of 1994–1995 and tree trunks were treated with a chemical to avoid re-sprouting in three sites (named: Bugac, Fülöpháza, Izsák). Mowing and hay removal were applied afterwards to help the recovery of the sandy grassland between 1995 and 2001. The experimental design consisted of six treatment and six control plots (10 m x 10 m each). The monitoring of the vegetation took place yearly in 1995–1999 and less frequently between 2002 and 2019 in 2 m by 2 m permanent sampling units (n = 18 per treatment; Reis et al. 2021 ). The other two experiments focused on the restoration of abandoned croplands in Fülöpháza. In the second experiment (EXP2), we manipulated soil nitrogen availability on three fields (named: Depression, Hummock, Meadow), abandoned 3–7 years prior to the experiment, in order to facilitate the establishment of the species of low-productive sandy grasslands. Carbon amendment (sugar and wood chips) was applied without soil disturbance between 1998 and 2003 (Halassy et al. 2016 ). The design of the experimental plots was the same as in the previous experiment (six treatment and six control plots, 10 m x 10 m each). We monitored the changes in the vegetation yearly between 1998 and 2004, and later in 2006, 2008, 2010 and 2018 (n = 18 sampling units per treatment) (Török et al. 2014 , Halassy et al. 2021 ). In the third experiment (EXP3), we combined the previous treatments (mowing and carbon amendment) with sowing on two arable fields abandoned in different years (named: Medium and Old). Plowing and harrowing were used as a pre-treatment to disrupt existing vegetation in the study areas (20 m by 20 m), followed by sowing, mowing or carbon amendment between 2002 and 2008. The experimental design consisted of eight replicates of 1 m x 1 m for each of the eight types of treatments per site separated by 1 m paths between the plots. Treatments were applied in these one square meter plots. Mowing and carbon amendment frequencies were similar as in EXP1 and EXP2, but only sugar was applied as carbon source. As for seeding, five hand-collected open sandy grassland species were sown together ( Festuca vaginata [1.55 g/m 2 ]; Stipa borysthenica [1.05 g/m 2 – later 1.31 g/m 2 ]; Koeleria glauca (Schrad.) DC. [1.00 g/m 2 ]; Dianthus serotinus Waldst. & Kit., Euphorbia seguieriana Neck. [the two latter 0.20 g/m 2 ]) (Reis 2021 ). Monitoring took place between 2003 and 2008 in all treatment plots, and the experiment was resampled in 2019 (Halassy et al. 2016 , 2019 , Llumiquinga et al. 2021 , Reis et al. 2022 ). A schematic figure showing the distribution of treatment and control plots in each of the three experiments is shown in the supporting information (Figure S1 ). Monitoring of invasive species in the experimental sites Vascular plant cover was estimated twice a year in early and late summer, and in this paper, we focus on invasive alien species only. From these, we considered neophyte species (taxa that were introduced to a new region after 1500 AD; Preston et al. 2004 ) in the analysis, as archaeophytes (taxa that settled in an area before 1500 AD; Preston et al. 2004 ) cannot be clearly distinguished from native species in the region. Neophyte species were categorized based on the work of Balogh et al. ( 2004 ) and subdivided into annual and perennial life form groups (the latter also including woody species that did not have enough occurrence to be analyzed separately). We calculated the maximum relative cover of each neophyte species and the two life form groups within plot per year for further analyses. The summary of all neophyte species and life form groups sampled in the experiments are shown in Table S1 . Monitoring of invasive species in the surroundings of the experiments To evaluate the invasive propagule pressure in the surroundings of the experimental sites, we established eight 100-meter-long transects from the center of each experimental site towards the eight cardinal directions (N, S, E, W, NE, NW, SE, SW) in 2020–2021 (Figure S2 ). We recorded the number of shoots of each neophyte species in 100 adjacent 1 x 1 m plots along each transect (n = 100 per transect). An example of the resulting heat maps can be found in Figure S3. We used the cumulative sum of the number of shoots of invasive species within 100 m buffer as a proxy for the landscape invasive propagule pressure. Data analysis The statistical analyses were carried out using R Studio 2022.12.0.353 (R core team 2022 ). We developed two groups of models for our two main questions applying Generalized Linear Mixed Models using Template Model Builder using the “ glmmTMB ” package (Brooks et al. 2017 ). Firstly, we analyzed the changes in the relative cover of annual and perennial neophyte species according to treatments and time for the three experiments separately. Treatment was used as fixed effects with two levels (1 = treatment, 0 = control), time since the start of intervention was included as a continuous variable, and plot ID nested in sites was treated as random effects in the models. The cover of annual and perennial neophyte species was used as dependent variables in separate models, both of them were square root-transformed and centered to meet assumptions of normality and homoscedasticity of residuals checked by the “ DHARMa ” package (Hartig 2020 ). To investigate the impact of treatment, time elapsed since treatment, and landscape invasive propagule pressure on the neophyte cover in the experimental areas, the three experiments were analyzed together. The dependent variables were also the two life form groups (annual and perennial), but we calculated their effect sizes by subtracting the relative neophyte cover of the treated plots from the relative neophyte cover of the control plots as a measure of restoration success. We applied the Hedges'g procedure to calculate the effect size due to the low number of samples (Hedges and Olkin 1985 ). From the fixed effects, treatment (with three levels: carbon amendment, mowing, seeding) was a categorical variable, while time elapsed since treatment and landscape invasive propagule pressure were continuous variables. The site variable was used as random effect. Normality and variance homogeneity of residuals were checked by the “ DHARMa ” package (Hartig 2020 ). In case of significant ( p < 0.05) treatment impacts, as a post hoc test, we used Estimated marginal means (Least-squares means) “ emmeans ” package’s Tukey pairwise comparison analysis function (Lenth 2023 ). Results Changes in annual and perennial neophyte cover over the years in the restoration experiments In case of the first experiment where treatment involved mowing at clear-cut black locust sites, only treatment (mowing) had a significant positive impact (χ 2 = 8.8411; Df = 1; p = 0.0029) on annual neophyte cover, and we found no significant impact of time or their interaction (Table S2 ). The relative cover of annual neophyte species increased in the first three years of treatment applications with the highest value found in the 3rd year since management (1997) in treatment plots (an average relative cover of 34.8%), but decreased later with some fluctuations to 0.9% in the last monitoring year (Fig. 2 A). In the case of perennial neophyte cover, only the elapsed time since the start of restoration intervention had a significant (χ 2 = 64.3666; Df = 1; p = < 0.001) impact, and treatment and the interaction did not (Table S2 ). The relative cover of perennial neophyte species increased with time (Fig. 2 B). The highest average relative cover was in last monitored year (2019) in control plots (48.7%), while the lowest was observed in the initial year in the control plots with 0.5%. Treatment plots had generally higher cover of perennial neophyte species than control plots, except in the last monitoring year, but these differences were not confirmed statistically. In the second experiment where carbon amendment was applied to accelerate grassland recovery on abandoned croplands, neither time nor treatment had a significant impact on the annual neophyte cover (Table S2 ). The relative cover of annual neophyte species showed some fluctuations: decreased initially during the treatment period, but returned to previous values by the 9th monitoring year (2006) and decreased again by the last survey (Fig. 2 C). The highest average annual relative cover was observed in the first year (1998) (in the control plots), which was 15.5%, while the lowest was observed in the 6th monitoring year (2003) in the treatment plots with only 0.7%. In the case of perennial neophyte cover, time and treatment did not have a significant impact, but their interaction did (χ 2 = 11.8595; Df = 1; p = 0.0005, Table S2 ). The average cover of perennial neophyte species increased with time, and reached 11% in the last year since management (2018) in treatment plots, while the lowest value was observed in the initial year in the treatment plots with 0.01% (Fig. 2 D). In the third experiment that included carbon amendment, mowing, and seeding on abandoned cropland, both treatment (χ 2 = 3.991; Df = 1; p = 0.0457) and time (χ 2 = 75.114; Df = 1; p = < 0.001) had a significant effect on annual neophytes without interaction (Table S2 ). In this case, the relative cover of annual neophytes was higher in the control plots. The relative cover of annual neophyte species clearly decreased with time (Fig. 2 E). The highest average relative cover of annual neophyte species was observed in the initial year (2003) in treatment plots (37.3%). We found the lowest average relative annual neophyte cover in the last monitoring year (2019) with only 1.32% in treatment plots. In the case of perennial neophyte species, neither treatment nor time or their interaction showed significant impact. The relative cover for perennial neophyte species was very low during the monitored years. Basically, they were absent in control plots and were insignificant in treatment plots in the beginning, and then increased in cover up to 2.4% in the control plots by the last year (2019) since the start of the management (Fig. 2 F). Effect of treatment, time and surrounding invasive propagule pressure on within site invasion When analyzed all experiments together, we found a significant impact of only the treatment in the case of annual species (χ2 = 30.8648; Df = 2; p = < 0.001), but no effect of time or invasive propagule pressure (Table S3). Tukey pairwise comparisons showed that seeding resulted in a significantly higher effect size than carbon amendment or mowing (Table S4) that shows a high positive impact on the progress of restoration indicated by a lower cover of annual neophytes in treated plots compared to control (Fig. 3 ). Carbon amendment and mowing were not statistically different from each other, and the effect sizes were around zero showing that the cover of annual neophyte species in treatment plots did not differ from control plots. In the case of perennial neophytes, neither treatment nor invasive propagule pressure affected the cover of perennial neophytes, but the elapsed time since interventions was significant (Table S3). Effect sizes started around zero at the beginning of treatments showing that treatment and control plots had similar cover of perennial neophytes initially (Figure S4). However, as the succession proceeded, effect sizes became negative, indicating that treatment plots harbored higher cover of perennial neophyte species than control plots with time with a few exceptions. All the effect size values can be found in Supplementary Information 2. Discussion Long-term trends of annual and perennial invasive species in sand grassland restoration Based on our research, the relative cover of annual invasive species has fluctuated, but showed an overall downward trend at the studied sites. However, we observed mostly an increasing trend in the cover of perennial invasive species over the monitored years. Among the annual invasive alien species, Ambrosia artemisiifolia L. was the most dominant species, and it has already been observed that abundance of this species decreases as succession progresses (Kröel-Dulay et al. 2019 ). Disturbances, such as drought or soil disturbance, can temporarily re-increase the abundance of weed species, including invasive alien species (Orbán et al. 2021 , Krpán 2023 ). The increasing trend in perennial species in our case is mainly due to clonal Asclepias syriaca L., which can easily colonize and spread in heavily disturbed, degraded, and open habitats and survive under various environmental conditions and treatments (Kelemen et al. 2016 , Bakacsy and Bagi 2020 ). Treatment significantly affected the cover of annual invasive species in the first and the third experiments but in different directions. In the first experiment, restorative mowing after clear-cutting of black locust opened up space for colonization that was primarily exploited by invasive alien species (e.g., Conyza canadensis ), while in control plots shrub encroachment hindered both grassland recovery and the establishment of invasive alien species (Reis et al. 2021 ). In the third experiment, treatment that also comprised seeding of native species resulted in a decrease in annual invasive species (Reis et al. 2023 ). The second experiment applied carbon amendment to create suitable abiotic conditions for the low productive native sand grasslands, but similar to other studies (Perry et al. 2010 ), failed to prove its negative impact on invasive species (Halassy et al. 2021 ). Effect of elapsed time, restoration intervention and invasive propagule pressure on restoration success When analyzing the three experiments together to include the landscape impact, treatment had a significant impact only on annual invasive species, while perennial invasive species increased with time irrespective of treatments and no significant impact of the invasive propagule pressure within a 100-meter buffer was found. From the three treatments (carbon amendment, mowing, seeding) involved in our study, only seeding had a significant negative effect on annual invasive species, but mowing and carbon amendment were neutral. Dispersal barriers are a major problem in the restoration of degraded habitats, which often need to be overcome by species introductions and assisted dispersal (Török et al. 2018 ). Moreover, providing priority to native species by sowing before the establishment of invasive alien species was found to be an important mechanism for increasing invasion resistance (Halassy et al. 2023 ). Sowing a seed mixture of native species can greatly reduce annual invasive species (Urza et al. 2019 , Halassy et al. 2023 ), also confirmed by our results, but we did not find a controlling effect on perennial invasive species. Perennial invasive species were not impacted by treatments, but increased with time during the succession process. In our case, the most dominant perennial invasive species was a clonal forb A. syriaca . What makes this invasive species so challenging and difficult to manage is its intensive vegetative spread, abundant fruit set and viable seed production (Gudžinskas et al. 2021 ). To our knowledge, A. syriaca does not respond significantly to a single treatment method (Bakacsy and Bagi 2020 ). It can recover from rhizomes after cutting or even chemical treatment, and the rhizome fragments left in the soil make it impossible to eradicate. Repeated cutting treatments are more promising, but we lack the knowledge on longer-term management effects and what happens if treatments are stopped (Berki et al. 2023 ). However, repeated treatments can be very costly and some treatments may even lead to further invasion (Valliere et al. 2019 ), so the options for controlling clonal perennial species are limited for now. We failed to prove the direct impact of invasive propagule pressure in the neighboring landscape. The degree of invasion depends on the inherent properties of habitats (invasibility), the invasive potential of the alien species, the actual propagule pressure and the landscape structure (Catford et al. 2011 , Vilà and Ibáñez 2011 , Guido et al. 2016, Kröel-Dulay et al. 2019 ). High invasive propagule pressure from the landscape can threaten the success of restoration efforts (Holl and Aide 2011 ). Although our previous research (Reis et al. 2022 ) has shown that habitat-level invasion in a 500 m buffer has a significant effect on the invasion of restoration sites, we failed to detect the direct effect of invasive propagule pressure at a shorter distance of 100 m. The lack of effect may be interpreted as a sign of saturation at the investigated scale (100 m), when a higher abundance of invasive species does not increase their cover in the restored plots (Vilá et al. 2011). Our contradicting results on the landscape effects at the two scales are also in line with findings that habitat type that reflects the combination of local abiotic conditions, current and historical land use and disturbances is the most important determinant of the level of invasion in the region (Csecserits et al. 2016 ). Conclusions Our research confirms that annual species may decline in the course of sand grassland restoration over time, but that aggressive, perennial invasive alien species (such as clonal A. syriaca ) may become more abundant on the long term once they become established, irrespective of treatments. Restorative treatments, especially sowing native species can accelerate the diminishing of invasive species, but only in case of annual invaders. Neither sowing, nor mowing or carbon amendment could sufficiently reduce the amount of perennial invasive species of sand grasslands. Mowing should be applied with care, because if mowing creates establishment windows, these can be exploited by invasive species if present in the landscape. The pressure of invasive alien species within 100 meters did not significantly affect the success of restoration efforts, despite the effect from a larger landscape buffer (Reis et al. 2022 ). Further research is needed in particular to identify methods to prevent the establishment and spread of perennial invasive species in restoration, since after establishment, adequate eradication methods are often lacking. Declarations Funding This work was supported by the National Research, Development and Innovation Office (NKFIH FK127996) and National Laboratory of Health Security (RRF-2.3.1-21-2022-00006) in Hungary. BPR was supported by FCT - Fundação para a Ciência e a Tecnologia (UIDB/00329/2020; PTDC/ASP-SIL/7743/2020) and both BPR and MV were financed by ELKH – Eötvös Loránd Research Network (ELKH SA-66/2021). EC was supported by the Ministry of Culture and Innovation (NTP-NFTÖ-22-B-0056 grants). MH was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences [grant number Bo/00145/23/8]. Competing interests: The author(s) declare none. Author Contributions Katalin Török and Melinda Halassy designed the experiments. Melinda Halassy and Nóra Sáradi conceived the study. Melinda Halassy, Nóra Sáradi, Bruna Paolinelli Reis, Edina Csákvári, Csonka Anna Cseperke, Máron Vörös, and Krisztina Neumann Verebényiné participated in field work and data sampling. Nóra Sáradi analyzed the data with inputs from Bruna Paolinelli Reis and Máron Vörös. Melinda Halassy and Nóra Sáradi wrote the article. All authors reviewed and commented on the manuscript. References Axmanová I, Kalusová V, Danihelka J, Dengler J, Pergl P, Pyšek P, Večeřa M, Attore F, Biurrun I, Boch S, Conradi T, Gavilán RG, Jimenéz-Alfaro B, Knollová I, Kuzemko A, Lenoir J, Leostrin A, Medvecká J, Moeslund JE, Obratov-Petkovic D, Svenning J-C, Tsiripidis I, Vassilev K, Chytrý M (2021) Neophyte invasions in European grasslands. Journal of Vegetation Science, 32:e12994. https://doi.org/10.1111/jvs.12994 Bagi I (1990) The vegetation map of the Szappan-szék Unesco Biosphere Reserve core area, Kiskunság National Park Hungary, Acta biologica , 36. 27-42. 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10:00:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4435901/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4435901/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10530-025-03613-5","type":"published","date":"2025-06-15T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58558645,"identity":"c4dde639-82e4-463b-924f-3f2be34ff742","added_by":"auto","created_at":"2024-06-18 08:37:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1775821,"visible":true,"origin":"","legend":"\u003cp\u003eThe location of the studied experiments (EXP 1, EXP 2, EXP 3) and their 100 m radius buffer in Bugac, Fülöpháza and Izsák in the Kiskunság region, Hungary, Europe. Projected coordinate system: EOV-1972. Maps made in QGIS, orthophotos by the Lechner Knowledge Center, Budapest, 2019.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4435901/v1/807276f06882205e1e17903a.png"},{"id":58559352,"identity":"dd6f02e8-87f3-4d4f-bd50-e3948d8f8873","added_by":"auto","created_at":"2024-06-18 08:45:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89325,"visible":true,"origin":"","legend":"\u003cp\u003eRelative cover of annual (A, C, E) and perennial (B, D, F) neophyte life form groups in treatment (red) and control (blue) plots within the three studied restoration experiments (boxplots are made in R Studio).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4435901/v1/5340e07ae999a5c9eae798f6.png"},{"id":58558640,"identity":"c5524596-6302-45b3-947f-dae509d1493c","added_by":"auto","created_at":"2024-06-18 08:37:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9848,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of carbon amendment, mowing and seeding on the effect size of annual neophyte species based on “\u003cem\u003eemmeans\u003c/em\u003e” Tukey pairwise comparison post hoc test. Positive values of effect size indicate lower cover of annual neophyte species in treatment than in control. Significant differences between treatments are indicated by lower case letters (boxplots are made in R Studio).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4435901/v1/8bebaa6e1bc312d46e7da0f9.png"},{"id":84726788,"identity":"6f0822dd-f08e-4896-af46-3afde481cad6","added_by":"auto","created_at":"2025-06-16 16:08:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2902867,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4435901/v1/06441114-5c70-4b2f-bebe-abba6e449944.pdf"},{"id":58559353,"identity":"12a96b26-8b2a-4bf4-bfe0-fc7bd93daef1","added_by":"auto","created_at":"2024-06-18 08:45:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2592578,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4435901/v1/a78615692b0dba299052a72c.docx"},{"id":58558643,"identity":"e0c991c3-cdad-4973-b486-e9e80c861c11","added_by":"auto","created_at":"2024-06-18 08:37:16","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15830,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4435901/v1/deec43c3552e88740959ff41.xlsx"}],"financialInterests":"","formattedTitle":"Restorative seeding controls annual invasive species, but perennials can thrive in the long term despite treatments in sand grassland restoration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOne of the main causes of biodiversity loss is the presence and spread of invasive alien species (IPBES 2023). In most cases, the removal of invasive alien species is not followed by the recovery of degraded native habitats, therefore ecological restoration is considered increasingly vital in the fight against invasion (Pearson et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEcological restoration aims to help restore degraded, damaged or destroyed ecosystems and has proven to be suitable for improving the ecological integrity of degraded habitats (Martin \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In some instances, revegetation efforts can successfully suppress invasive plants (Halassy et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, restorative interventions can also lead to disturbances. Soil disturbance, burning, grazing, mowing and weed control are common restoration interventions which create open spaces in a community and increase the resources available to native species (Hobbs et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Flory and Clay \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Papanastasis \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, these types of disturbances can also mediate invasions (Hobbs and Huenneke \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), especially when the invaders are disturbance-adapted species with good dispersal capacities (Funk et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, in search of best restoration practices, we need to understand how invasive species respond to restorative treatments on the long term and how the presence of invasive species in the surrounding landscape influences the progress of restoration.\u003c/p\u003e \u003cp\u003eSome studies have shown that different plant life forms respond differently to restorative interventions, which should therefore also be taken into consideration in management and restoration (Nyamai et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Halassy et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Annual invasive species have a special set of traits that help them to successfully colonize new areas: they are characterized by efficient reproduction and dispersal, i.e. a wide variety of mixed dispersal vectors, and an appropriate germination strategy (e.g. biotic conditions are sensed by the seeds) can ensure their quick establishment in a new habitat (Fenesi \u0026amp; Botta-Duk\u0026aacute;t \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In contrast, for perennial invasive species, population expansion and adaptive evolution to new habitats can be much slower and more difficult, but longer residence times allow perennial invasive species to spread to more places, increasing the scale of invasion (Ni et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, the two life form groups might require different approaches for nature conservation and restoration of the native habitat. For example, Ramula and colleagues (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) found that manipulation of growth and reproductive mechanisms in annual invasive plants always resulted in population declines. However, in the case of perennial invasive species, neither influencing survival, growth nor reproductive mechanism separately was sufficient to reduce the population, while influencing survival and growth or survival and reproduction mechanisms together were more effective.\u003c/p\u003e \u003cp\u003eVarious forms of active restoration methods exist to mitigate the impact of invasive species and improve biodiversity (Weidlich et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Mowing, one of the widely used restoration interventions has been shown to be efficient in reducing the density of perennial invasive plants even in the short term (Nagy et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and it also strongly hinders the reproductive success of annual invasive species (Milakovic et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Nevertheless, mowing and grazing can open up windows for colonization, which invasive species with good dispersal capacity can exploit first (Reis et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Seeding has also been shown to be effective in reducing annual invasive species (Urza et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and affecting the biomass production of perennial invasive species (Reinhardt and Galatowitsch \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, indirect methods, such as soil nitrogen immobilization using carbon sources, have been less successful in controlling invasive species (Perry et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and have been reported to affect mainly annual invasive species (Davis et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLandscape factors can further modify the impact of restorative measures, therefore assessing and understanding landscape-scale impacts is essential for planning successful restoration interventions (Helsen et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Prach et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Several studies have shown that the most important landscape features that influence the success of a restoration intervention are the area and perimeter of semi-natural habitat patches in the landscape and the proximity of propagule sources (Helsen et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Guido et al. 2016). This combined with high propagule pressure is one of the main factors influencing invasion (Catford et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Kr\u0026ouml;el-Dulay et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). There are habitats rich in native species that are optimal sources of natural recovery. However, in an anthropogenised landscape, many habitats are the sources of invasive species that can undermine restoration efforts (Holl and Aide \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Vil\u0026agrave; and Ib\u0026aacute;\u0026ntilde;ez \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Csecserits et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Guido et al. 2016).\u003c/p\u003e \u003cp\u003eGrasslands are one of the most vulnerable habitats as they are subject to the highest levels of plant invasion (Catford and Jones \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The most abundant invasive alien species are terrestrial plants, which represent nearly half of all invasive species in Europe (6 368 species) (Costello et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Land-use change in general fosters plant invasions (Vil\u0026agrave; and Ib\u0026aacute;\u0026ntilde;ez \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and grasslands were found to become more prone to invasion when traditional human interventions (e.g. grazing or mowing) are abandoned (Axmanov\u0026aacute; et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). According to a countrywide survey (called M\u0026Eacute;TA), 5.5% of the territory of Hungary is covered by invasive perennial plants (not counting woody invaders) (Botta-Duk\u0026aacute;t \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The rate of land abandonment has accelerated in Hungary over the past three decades (Valk\u0026oacute; et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), creating opportunities for the regeneration of abandoned lands, but also for the advance of invasive species (T\u0026ouml;r\u0026ouml;k et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Not surprisingly, grassland restoration has been ranked among the top 50 conservation research priorities (Mih\u0026oacute;k et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and the control of invasive species is in the focus of restorative interventions (T\u0026ouml;r\u0026ouml;k et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMethods for the restoration of Pannonian sandy grasslands are studied for about 20 years (Kiskun Restoration Experiments, KISKUN LTER). We applied mowing, carbon amendment and seeding after the cessation of arable cultivation or the clear-cutting of non-native plantations and monitored the outcome of restorative interventions in the long term (Halassy et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Results so far show that the outcome of restoration interventions depends not only on the restorative intervention itself, but also on the elapsed time and the surrounding landscape (Reis et al \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although a landscape impact is visible, there is a lack of knowledge about the distances from which the pressure of invasive alien species may pose a threat to the restoration efforts and on the different behavior of annual and perennial invasive species. Building on our previous research (Reis et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), in which we described the impact of landscape habitat composition on the long-term success of restoration, we used a finer scale transect method to study the impact of invasive propagule pressure from the neighborhoods surrounding the restoration sites on invasive species abundance and separately analyzed the response of annual and perennial invasive species at the restoration sites.\u003c/p\u003e \u003cp\u003eThe questions of our research are: (1) How does the level of annual and perennial invasion change with time in three sand grassland restoration experiments on the long term? (2) To what extent does the degree of invasion within the restoration experiments depend on the type of restoration intervention, the time since the intervention and the invasive propagule pressure from the landscape?\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area\u003c/h2\u003e \u003cp\u003eWe collected long-term datasets from three different experiments, which were carried out at the Kiskun LTER site (Kiskun LTER 2005\u0026ndash;2024), Hungary. The experiments are near three settlements (Bugac: 46\u0026deg;39'N, 19\u0026deg;36\u0026rsquo;E; F\u0026uuml;l\u0026ouml;ph\u0026aacute;za: 46\u0026deg;52'N, 19\u0026deg;24'E; Izs\u0026aacute;k: 46\u0026deg;45'N, 19\u0026deg;19'E) in the Kiskuns\u0026aacute;g Sand Ridge (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The area lies in a harsh environment regarding the temperature, moisture, wind and soil conditions (Borhidi \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Kov\u0026aacute;cs-L\u0026aacute;ng et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The average annual temperature is 10.5\u0026deg;C with large daily and annual fluctuations. The yearly precipitation is 520\u0026ndash;540 mm, and drought events have become more frequent (especially during the summer). The predominant wind direction is NW to SE (Bagi \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). The soils are Calcaric Arenosol with more than 90% of sand and less than 1% humus content (Z\u0026oacute;lyomi et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Kov\u0026aacute;cs-L\u0026aacute;ng et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Buz\u0026aacute;si et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe area is situated in the forest-steppe zone (Erdős et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) with a mosaic of different types of communities depending on topography and the parent material. One of its most typical components at the driest locations is the open sandy grassland (\u003cem\u003eFestucetum vaginatae \u0026lsquo;danubiale\u0026rsquo;\u003c/em\u003e community\u003cem\u003e)\u003c/em\u003e. The \u003cem\u003eFestucetum vaginatae\u003c/em\u003e \u0026lsquo;\u003cem\u003edanubiale\u0026rsquo;\u003c/em\u003e is a semi-desert-like community, rich in endemic species and dominated by the tussock grasses \u003cem\u003eFestuca vaginata\u003c/em\u003e Waldst. \u0026amp; Kit. ex Willd. and \u003cem\u003eStipa borysthenica\u003c/em\u003e Klokov ex Prokudin, which is present on the tops and Southern slopes of nutrient-poor and coarse-textured calcareous sand dunes (Kov\u0026aacute;cs-L\u0026aacute;ng et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Other typical natural habitat types are closed sand steppe, poplar-juniper shrubland, open and closed natural forest, and wetlands (mesotrophic wet meadows, \u003cem\u003eMolinia\u003c/em\u003e-dominated meadows, and marshes) (Bir\u0026oacute; et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e). Due to historical human land transformation, most of the present landscape is covered by agricultural areas (57%), forests - mostly non-native plantations \u0026minus;\u0026thinsp;(19%), and settlements (6%) (CORINE 2000). Agricultural cultivation reached its peak during the socialist regime, in the 1960s. Afforestation by black and scots pine (\u003cem\u003ePinus nigra\u003c/em\u003e J.F.Arnold, \u003cem\u003ePinus sylvestris\u003c/em\u003e L.), black locust (\u003cem\u003eRobinia pseudoacacia\u003c/em\u003e L.\u003cem\u003e)\u003c/em\u003e and native white poplar (\u003cem\u003ePopolus alba\u003c/em\u003e L.) was carried out during the 19\u0026mdash;20th centuries to hold the sand. Land-use changes have led to the preservation of only approximately 19% of semi-natural habitats in the region. This includes not only remnants of primary habitats but also disturbed meadows and dry grasslands, some of which have been encroached by non-native trees (Bir\u0026oacute; et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring the post-socialist transformation (1987\u0026ndash;1999), the abandonment of agricultural land reached a significant level, especially in low productive areas, such as the Kiskuns\u0026aacute;g, and besides the spontaneous recovery of native vegetation, invasive alien species began to spread to these abandoned areas (Bir\u0026oacute; et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e; \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003eb\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRestoration experiments\u003c/h2\u003e \u003cp\u003eWe conducted several experiments in the region with the aim to assist the recovery of sandy grassland habitats in degraded areas, and here we analyzed the invasion of non-native annual and perennial plant species in three of them offering long-term data. In the first experiment (EXP1), afforestation with non-native \u003cem\u003eRobinia pseudoacacia\u003c/em\u003e caused grassland degradation. In order to restore grasslands, the trees were clear-cut in the winter of 1994\u0026ndash;1995 and tree trunks were treated with a chemical to avoid re-sprouting in three sites (named: Bugac, F\u0026uuml;l\u0026ouml;ph\u0026aacute;za, Izs\u0026aacute;k). Mowing and hay removal were applied afterwards to help the recovery of the sandy grassland between 1995 and 2001. The experimental design consisted of six treatment and six control plots (10 m x 10 m each). The monitoring of the vegetation took place yearly in 1995\u0026ndash;1999 and less frequently between 2002 and 2019 in 2 m by 2 m permanent sampling units (n\u0026thinsp;=\u0026thinsp;18 per treatment; Reis et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe other two experiments focused on the restoration of abandoned croplands in F\u0026uuml;l\u0026ouml;ph\u0026aacute;za. In the second experiment (EXP2), we manipulated soil nitrogen availability on three fields (named: Depression, Hummock, Meadow), abandoned 3\u0026ndash;7 years prior to the experiment, in order to facilitate the establishment of the species of low-productive sandy grasslands. Carbon amendment (sugar and wood chips) was applied without soil disturbance between 1998 and 2003 (Halassy et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The design of the experimental plots was the same as in the previous experiment (six treatment and six control plots, 10 m x 10 m each). We monitored the changes in the vegetation yearly between 1998 and 2004, and later in 2006, 2008, 2010 and 2018 (n\u0026thinsp;=\u0026thinsp;18 sampling units per treatment) (T\u0026ouml;r\u0026ouml;k et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Halassy et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the third experiment (EXP3), we combined the previous treatments (mowing and carbon amendment) with sowing on two arable fields abandoned in different years (named: Medium and Old). Plowing and harrowing were used as a pre-treatment to disrupt existing vegetation in the study areas (20 m by 20 m), followed by sowing, mowing or carbon amendment between 2002 and 2008. The experimental design consisted of eight replicates of 1 m x 1 m for each of the eight types of treatments per site separated by 1 m paths between the plots. Treatments were applied in these one square meter plots. Mowing and carbon amendment frequencies were similar as in EXP1 and EXP2, but only sugar was applied as carbon source. As for seeding, five hand-collected open sandy grassland species were sown together (\u003cem\u003eFestuca vaginata\u003c/em\u003e [1.55 g/m\u003csup\u003e2\u003c/sup\u003e]; \u003cem\u003eStipa borysthenica\u003c/em\u003e [1.05 g/m\u003csup\u003e2\u003c/sup\u003e \u0026ndash; later 1.31 g/m\u003csup\u003e2\u003c/sup\u003e]; \u003cem\u003eKoeleria glauca\u003c/em\u003e (Schrad.) DC. [1.00 g/m\u003csup\u003e2\u003c/sup\u003e]; \u003cem\u003eDianthus serotinus\u003c/em\u003e Waldst. \u0026amp; Kit., \u003cem\u003eEuphorbia seguieriana\u003c/em\u003e Neck. [the two latter 0.20 g/m\u003csup\u003e2\u003c/sup\u003e]) (Reis \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Monitoring took place between 2003 and 2008 in all treatment plots, and the experiment was resampled in 2019 (Halassy et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Llumiquinga et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Reis et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA schematic figure showing the distribution of treatment and control plots in each of the three experiments is shown in the supporting information (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMonitoring of invasive species in the experimental sites\u003c/h2\u003e \u003cp\u003eVascular plant cover was estimated twice a year in early and late summer, and in this paper, we focus on invasive alien species only. From these, we considered neophyte species (taxa that were introduced to a new region after 1500 AD; Preston et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) in the analysis, as archaeophytes (taxa that settled in an area before 1500 AD; Preston et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) cannot be clearly distinguished from native species in the region. Neophyte species were categorized based on the work of Balogh et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and subdivided into annual and perennial life form groups (the latter also including woody species that did not have enough occurrence to be analyzed separately). We calculated the maximum relative cover of each neophyte species and the two life form groups within plot per year for further analyses. The summary of all neophyte species and life form groups sampled in the experiments are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMonitoring of invasive species in the surroundings of the experiments\u003c/h2\u003e \u003cp\u003eTo evaluate the invasive propagule pressure in the surroundings of the experimental sites, we established eight 100-meter-long transects from the center of each experimental site towards the eight cardinal directions (N, S, E, W, NE, NW, SE, SW) in 2020\u0026ndash;2021 (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). We recorded the number of shoots of each neophyte species in 100 adjacent 1 x 1 m plots along each transect (n\u0026thinsp;=\u0026thinsp;100 per transect). An example of the resulting heat maps can be found in Figure S3. We used the cumulative sum of the number of shoots of invasive species within 100 m buffer as a proxy for the landscape invasive propagule pressure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe statistical analyses were carried out using R Studio 2022.12.0.353 (R core team \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We developed two groups of models for our two main questions applying Generalized Linear Mixed Models using Template Model Builder using the \u0026ldquo;\u003cem\u003eglmmTMB\u003c/em\u003e\u0026rdquo; package (Brooks et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Firstly, we analyzed the changes in the relative cover of annual and perennial neophyte species according to treatments and time for the three experiments separately. Treatment was used as fixed effects with two levels (1\u0026thinsp;=\u0026thinsp;treatment, 0\u0026thinsp;=\u0026thinsp;control), time since the start of intervention was included as a continuous variable, and plot ID nested in sites was treated as random effects in the models. The cover of annual and perennial neophyte species was used as dependent variables in separate models, both of them were square root-transformed and centered to meet assumptions of normality and homoscedasticity of residuals checked by the \u0026ldquo;\u003cem\u003eDHARMa\u003c/em\u003e\u0026rdquo; package (Hartig \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo investigate the impact of treatment, time elapsed since treatment, and landscape invasive propagule pressure on the neophyte cover in the experimental areas, the three experiments were analyzed together. The dependent variables were also the two life form groups (annual and perennial), but we calculated their effect sizes by subtracting the relative neophyte cover of the treated plots from the relative neophyte cover of the control plots as a measure of restoration success. We applied the Hedges'g procedure to calculate the effect size due to the low number of samples (Hedges and Olkin \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). From the fixed effects, treatment (with three levels: carbon amendment, mowing, seeding) was a categorical variable, while time elapsed since treatment and landscape invasive propagule pressure were continuous variables. The site variable was used as random effect. Normality and variance homogeneity of residuals were checked by the \u0026ldquo;\u003cem\u003eDHARMa\u003c/em\u003e\u0026rdquo; package (Hartig \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In case of significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) treatment impacts, as a post hoc test, we used Estimated marginal means (Least-squares means) \u0026ldquo;\u003cem\u003eemmeans\u003c/em\u003e\u0026rdquo; package\u0026rsquo;s Tukey pairwise comparison analysis function (Lenth \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eChanges in annual and perennial neophyte cover over the years in the restoration experiments\u003c/h2\u003e \u003cp\u003eIn case of the first experiment where treatment involved mowing at clear-cut black locust sites, only treatment (mowing) had a significant positive impact (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;8.8411; Df\u0026thinsp;=\u0026thinsp;1; p\u0026thinsp;=\u0026thinsp;0.0029) on annual neophyte cover, and we found no significant impact of time or their interaction (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The relative cover of annual neophyte species increased in the first three years of treatment applications with the highest value found in the 3rd year since management (1997) in treatment plots (an average relative cover of 34.8%), but decreased later with some fluctuations to 0.9% in the last monitoring year (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the case of perennial neophyte cover, only the elapsed time since the start of restoration intervention had a significant (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;64.3666; Df\u0026thinsp;=\u0026thinsp;1; p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001) impact, and treatment and the interaction did not (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The relative cover of perennial neophyte species increased with time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The highest average relative cover was in last monitored year (2019) in control plots (48.7%), while the lowest was observed in the initial year in the control plots with 0.5%. Treatment plots had generally higher cover of perennial neophyte species than control plots, except in the last monitoring year, but these differences were not confirmed statistically.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the second experiment where carbon amendment was applied to accelerate grassland recovery on abandoned croplands, neither time nor treatment had a significant impact on the annual neophyte cover (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The relative cover of annual neophyte species showed some fluctuations: decreased initially during the treatment period, but returned to previous values by the 9th monitoring year (2006) and decreased again by the last survey (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The highest average annual relative cover was observed in the first year (1998) (in the control plots), which was 15.5%, while the lowest was observed in the 6th monitoring year (2003) in the treatment plots with only 0.7%. In the case of perennial neophyte cover, time and treatment did not have a significant impact, but their interaction did (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;11.8595; Df\u0026thinsp;=\u0026thinsp;1; p\u0026thinsp;=\u0026thinsp;0.0005, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The average cover of perennial neophyte species increased with time, and reached 11% in the last year since management (2018) in treatment plots, while the lowest value was observed in the initial year in the treatment plots with 0.01% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eIn the third experiment that included carbon amendment, mowing, and seeding on abandoned cropland, both treatment (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;3.991; Df\u0026thinsp;=\u0026thinsp;1; p\u0026thinsp;=\u0026thinsp;0.0457) and time (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;75.114; Df\u0026thinsp;=\u0026thinsp;1; p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001) had a significant effect on annual neophytes without interaction (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In this case, the relative cover of annual neophytes was higher in the control plots. The relative cover of annual neophyte species clearly decreased with time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The highest average relative cover of annual neophyte species was observed in the initial year (2003) in treatment plots (37.3%). We found the lowest average relative annual neophyte cover in the last monitoring year (2019) with only 1.32% in treatment plots. In the case of perennial neophyte species, neither treatment nor time or their interaction showed significant impact. The relative cover for perennial neophyte species was very low during the monitored years. Basically, they were absent in control plots and were insignificant in treatment plots in the beginning, and then increased in cover up to 2.4% in the control plots by the last year (2019) since the start of the management (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEffect of treatment, time and surrounding invasive propagule pressure on within site invasion\u003c/h2\u003e \u003cp\u003eWhen analyzed all experiments together, we found a significant impact of only the treatment in the case of annual species (χ2\u0026thinsp;=\u0026thinsp;30.8648; Df\u0026thinsp;=\u0026thinsp;2; p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but no effect of time or invasive propagule pressure (Table S3). Tukey pairwise comparisons showed that seeding resulted in a significantly higher effect size than carbon amendment or mowing (Table S4) that shows a high positive impact on the progress of restoration indicated by a lower cover of annual neophytes in treated plots compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Carbon amendment and mowing were not statistically different from each other, and the effect sizes were around zero showing that the cover of annual neophyte species in treatment plots did not differ from control plots. In the case of perennial neophytes, neither treatment nor invasive propagule pressure affected the cover of perennial neophytes, but the elapsed time since interventions was significant (Table S3). Effect sizes started around zero at the beginning of treatments showing that treatment and control plots had similar cover of perennial neophytes initially (Figure S4). However, as the succession proceeded, effect sizes became negative, indicating that treatment plots harbored higher cover of perennial neophyte species than control plots with time with a few exceptions. All the effect size values can be found in Supplementary Information 2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLong-term trends of annual and perennial invasive species in sand grassland restoration\u003c/h2\u003e \u003cp\u003eBased on our research, the relative cover of annual invasive species has fluctuated, but showed an overall downward trend at the studied sites. However, we observed mostly an increasing trend in the cover of perennial invasive species over the monitored years. Among the annual invasive alien species, \u003cem\u003eAmbrosia artemisiifolia\u003c/em\u003e L. was the most dominant species, and it has already been observed that abundance of this species decreases as succession progresses (Kr\u0026ouml;el-Dulay et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Disturbances, such as drought or soil disturbance, can temporarily re-increase the abundance of weed species, including invasive alien species (Orb\u0026aacute;n et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Krp\u0026aacute;n \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The increasing trend in perennial species in our case is mainly due to clonal \u003cem\u003eAsclepias syriaca\u003c/em\u003e L., which can easily colonize and spread in heavily disturbed, degraded, and open habitats and survive under various environmental conditions and treatments (Kelemen et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Bakacsy and Bagi \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTreatment significantly affected the cover of annual invasive species in the first and the third experiments but in different directions. In the first experiment, restorative mowing after clear-cutting of black locust opened up space for colonization that was primarily exploited by invasive alien species (e.g., \u003cem\u003eConyza canadensis\u003c/em\u003e), while in control plots shrub encroachment hindered both grassland recovery and the establishment of invasive alien species (Reis et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the third experiment, treatment that also comprised seeding of native species resulted in a decrease in annual invasive species (Reis et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The second experiment applied carbon amendment to create suitable abiotic conditions for the low productive native sand grasslands, but similar to other studies (Perry et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), failed to prove its negative impact on invasive species (Halassy et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffect of elapsed time, restoration intervention and invasive propagule pressure on restoration success\u003c/h2\u003e \u003cp\u003eWhen analyzing the three experiments together to include the landscape impact, treatment had a significant impact only on annual invasive species, while perennial invasive species increased with time irrespective of treatments and no significant impact of the invasive propagule pressure within a 100-meter buffer was found.\u003c/p\u003e \u003cp\u003eFrom the three treatments (carbon amendment, mowing, seeding) involved in our study, only seeding had a significant negative effect on annual invasive species, but mowing and carbon amendment were neutral. Dispersal barriers are a major problem in the restoration of degraded habitats, which often need to be overcome by species introductions and assisted dispersal (T\u0026ouml;r\u0026ouml;k et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, providing priority to native species by sowing before the establishment of invasive alien species was found to be an important mechanism for increasing invasion resistance (Halassy et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Sowing a seed mixture of native species can greatly reduce annual invasive species (Urza et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Halassy et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), also confirmed by our results, but we did not find a controlling effect on perennial invasive species.\u003c/p\u003e \u003cp\u003ePerennial invasive species were not impacted by treatments, but increased with time during the succession process. In our case, the most dominant perennial invasive species was a clonal forb \u003cem\u003eA. syriaca\u003c/em\u003e. What makes this invasive species so challenging and difficult to manage is its intensive vegetative spread, abundant fruit set and viable seed production (Gudžinskas et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To our knowledge, \u003cem\u003eA. syriaca\u003c/em\u003e does not respond significantly to a single treatment method (Bakacsy and Bagi \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It can recover from rhizomes after cutting or even chemical treatment, and the rhizome fragments left in the soil make it impossible to eradicate. Repeated cutting treatments are more promising, but we lack the knowledge on longer-term management effects and what happens if treatments are stopped (Berki et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, repeated treatments can be very costly and some treatments may even lead to further invasion (Valliere et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), so the options for controlling clonal perennial species are limited for now.\u003c/p\u003e \u003cp\u003eWe failed to prove the direct impact of invasive propagule pressure in the neighboring landscape. The degree of invasion depends on the inherent properties of habitats (invasibility), the invasive potential of the alien species, the actual propagule pressure and the landscape structure (Catford et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Vil\u0026agrave; and Ib\u0026aacute;\u0026ntilde;ez \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Guido et al. 2016, Kr\u0026ouml;el-Dulay et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). High invasive propagule pressure from the landscape can threaten the success of restoration efforts (Holl and Aide \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Although our previous research (Reis et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) has shown that habitat-level invasion in a 500 m buffer has a significant effect on the invasion of restoration sites, we failed to detect the direct effect of invasive propagule pressure at a shorter distance of 100 m. The lack of effect may be interpreted as a sign of saturation at the investigated scale (100 m), when a higher abundance of invasive species does not increase their cover in the restored plots (Vil\u0026aacute; et al. 2011). Our contradicting results on the landscape effects at the two scales are also in line with findings that habitat type that reflects the combination of local abiotic conditions, current and historical land use and disturbances is the most important determinant of the level of invasion in the region (Csecserits et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur research confirms that annual species may decline in the course of sand grassland restoration over time, but that aggressive, perennial invasive alien species (such as clonal \u003cem\u003eA. syriaca\u003c/em\u003e) may become more abundant on the long term once they become established, irrespective of treatments. Restorative treatments, especially sowing native species can accelerate the diminishing of invasive species, but only in case of annual invaders. Neither sowing, nor mowing or carbon amendment could sufficiently reduce the amount of perennial invasive species of sand grasslands. Mowing should be applied with care, because if mowing creates establishment windows, these can be exploited by invasive species if present in the landscape. The pressure of invasive alien species within 100 meters did not significantly affect the success of restoration efforts, despite the effect from a larger landscape buffer (Reis et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Further research is needed in particular to identify methods to prevent the establishment and spread of perennial invasive species in restoration, since after establishment, adequate eradication methods are often lacking.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research, Development and Innovation Office (NKFIH FK127996) and National Laboratory of Health Security (RRF-2.3.1-21-2022-00006) in Hungary. \u0026nbsp; BPR was supported by FCT - Funda\u0026ccedil;\u0026atilde;o para a Ci\u0026ecirc;ncia e a Tecnologia (UIDB/00329/2020; PTDC/ASP-SIL/7743/2020) and both BPR and MV were financed by ELKH \u0026ndash; E\u0026ouml;tv\u0026ouml;s Lor\u0026aacute;nd Research Network (ELKH SA-66/2021). EC was supported by the Ministry of Culture and Innovation (NTP-NFT\u0026Ouml;-22-B-0056 grants). MH was supported by the J\u0026aacute;nos Bolyai Research Scholarship of the Hungarian Academy of Sciences [grant number Bo/00145/23/8].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The author(s) declare none.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKatalin T\u0026ouml;r\u0026ouml;k and Melinda Halassy designed the experiments. Melinda Halassy and N\u0026oacute;ra S\u0026aacute;radi conceived the study. Melinda Halassy, N\u0026oacute;ra S\u0026aacute;radi, Bruna Paolinelli Reis, Edina Cs\u0026aacute;kv\u0026aacute;ri, Csonka Anna Cseperke, M\u0026aacute;ron V\u0026ouml;r\u0026ouml;s, and Krisztina Neumann Vereb\u0026eacute;nyin\u0026eacute; participated in field work and data sampling. N\u0026oacute;ra S\u0026aacute;radi analyzed the data with inputs from Bruna Paolinelli Reis and M\u0026aacute;ron V\u0026ouml;r\u0026ouml;s. Melinda Halassy and N\u0026oacute;ra S\u0026aacute;radi wrote the article. All authors reviewed and commented on the manuscript.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAxmanov\u0026aacute; I, Kalusov\u0026aacute; V, Danihelka J, Dengler J, Pergl P, Py\u0026scaron;ek P, Večeřa M, Attore F, Biurrun I, Boch S, Conradi T, Gavil\u0026aacute;n RG, Jimen\u0026eacute;z-Alfaro B, Knollov\u0026aacute; I, Kuzemko A, Lenoir J, Leostrin A, Medveck\u0026aacute; J, Moeslund JE, Obratov-Petkovic D, Svenning J-C, Tsiripidis I, Vassilev K, Chytr\u0026yacute; M (2021) Neophyte invasions in European grasslands. \u003cem\u003eJournal of Vegetation Science,\u003c/em\u003e 32:e12994. https://doi.org/10.1111/jvs.12994\u003c/li\u003e\n\u003cli\u003eBagi I (1990) The vegetation map of the Szappan-sz\u0026eacute;k Unesco Biosphere Reserve core area, Kiskuns\u0026aacute;g National Park Hungary, \u003cem\u003eActa biologica\u003c/em\u003e, 36. 27-42.\u003c/li\u003e\n\u003cli\u003eBakacsy L, Bagi I (2020) Survival and regeneration ability of clonal common milkweed (\u003cem\u003eAsclepias syriaca\u003c/em\u003e L.) after a single herbicide treatment in natural open sand grasslands. \u003cem\u003eScientific Reports,\u003c/em\u003e 10, 14222 https://doi.org/10.1038/s41598-020-71202-8\u003c/li\u003e\n\u003cli\u003eBalogh L, Dancza I, Kir\u0026aacute;ly G (2004) A magyarorsz\u0026aacute;gi neofitonok időszerű jegyz\u0026eacute;ke \u0026eacute;s besorol\u0026aacute;suk inv\u0026aacute;zi\u0026oacute;s szempontb\u0026oacute;l. 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Journal of Applied Ecology,\u003c/em\u003e 57: 1806\u0026ndash;1817. http://dx.doi.org/10.1111/1365-2664.13656\u003c/li\u003e\n\u003cli\u003eZ\u0026oacute;lyomi B, K\u0026eacute;ri M, Horv\u0026aacute;th F (1997) Spatial and temporal changes in the frequency of climatic year types in the Carpathian Basin. \u003cem\u003eCoenoses\u003c/em\u003e 12:33-41.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biological-invasions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"binv","sideBox":"Learn more about [Biological Invasions](https://www.springer.com/journal/10530)","snPcode":"10530","submissionUrl":"https://submission.nature.com/new-submission/10530/3","title":"Biological Invasions","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"dry grassland restoration, invasive alien plant species, invasive propagule pressure, landscape impact, long-term monitoring","lastPublishedDoi":"10.21203/rs.3.rs-4435901/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4435901/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiodiversity loss caused by invasive alien species is a major problem in planetary perspective. Ecological restoration is an important tool to counteract invasions, but invasive species may negatively affect restoration if present in the landscape. We investigated long-term changes in annual and perennial invasive alien species abundance in three sandy grassland restoration experiments. We evaluated the dependence of annual and perennial invasive species abundance on initial restoration intervention, invasive species propagule pressure from the surrounding landscape, and time since interventions. Restoration interventions (seeding, mowing and carbon amendment) were conducted at a total of eight sites in the Kiskuns\u0026aacute;g region of Hungary. The interventions took place between 1995 and 2003 and were monitored for 17\u0026ndash;25 years. To assess invasive propagule pressure around the experimental sites, total shoot numbers in adjacent 1 m by 1 m plots along 100-meter-long transects were counted in 2020\u0026ndash;2021 from the center of the eight experimental sites. Invasive propagule pressure within a 100-meter buffer did not explain changes in the abundance of annual and perennial invasive species. The cover of annual invasive species has mostly decreased over time, and treatment (mainly seeding) could accelerate this process. The cover of perennial invasive species increased over time irrespective of applied treatments and landscape invasive propagule pressure. Our research showed that seeding with native species is an effective tool for restoring sandy grasslands and preventing the spread of annual invasive species, but our toolbox for preventing perennial invasion in grassland restoration is limited.\u003c/p\u003e","manuscriptTitle":"Restorative seeding controls annual invasive species, but perennials can thrive in the long term despite treatments in sand grassland restoration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-18 08:37:11","doi":"10.21203/rs.3.rs-4435901/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-06-11T15:39:27+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-04T20:40:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Biological Invasions","date":"2024-05-25T11:51:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-21T12:32:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Invasions","date":"2024-05-17T06:00:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-invasions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"binv","sideBox":"Learn more about [Biological Invasions](https://www.springer.com/journal/10530)","snPcode":"10530","submissionUrl":"https://submission.nature.com/new-submission/10530/3","title":"Biological Invasions","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c1d92c9a-66d8-427d-9887-93b5cfe0bf7a","owner":[],"postedDate":"June 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T16:05:41+00:00","versionOfRecord":{"articleIdentity":"rs-4435901","link":"https://doi.org/10.1007/s10530-025-03613-5","journal":{"identity":"biological-invasions","isVorOnly":false,"title":"Biological Invasions"},"publishedOn":"2025-06-15 15:57:16","publishedOnDateReadable":"June 15th, 2025"},"versionCreatedAt":"2024-06-18 08:37:11","video":"","vorDoi":"10.1007/s10530-025-03613-5","vorDoiUrl":"https://doi.org/10.1007/s10530-025-03613-5","workflowStages":[]},"version":"v1","identity":"rs-4435901","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4435901","identity":"rs-4435901","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}