Plant-Soil Interactions of an Invasive Plant Species in its Native Range Help to Explain its Invasion Success Elsewhere


 Purpose: To compare plant-soil feedback (PSF) of invasive Cirsium vulgare and non-invasive C. oleraceum in their native range to test a hypothesis that the invasive species is more limited by specialized pathogens in the native range and/or able to benefit more from generalist mutualists, and thus may benefit more from loss of specialized soil biota in a secondary range.Methods: We assessed changes in soil nutrients and biota following soil conditioning by each species and compared performance of plants grown in self-conditioned and control soil, from which all, some or no biota was excluded. Results: The invasive species depleted more nutrients than the non-invasive species and coped better with altered nutrient levels. The invasive species had higher seedling emergence which benefited from presence of non-specific microbes. The invasive species biomass responded less positively to specialized (self-conditioned) microbiota and more negatively to specialized larger-sized biota compared to the non-specialized control biota, suggesting the species may benefit more from enemy release and suffer less from loss of specialized mutualists when introduced to a secondary range. The invasive species showed greater ability to decrease its root-shoot ratio in presence of harmful biota and thus reduce their negative effects on its performance.Conclusions: Our study highlights the utility of detailed PSF research in the native range of species for understanding the factors that regulate performance of invasive and non-invasive species in their native range, and for pinpointing the types of biota involved in their regulation and how this changes across the plants life cycle.


Introduction
Understanding the success of invasive species and why some alien plants become invasive while others fail is a fundamental goal in the eld of invasion ecology. Despite a high number of hypotheses explaining the success of invasive species, such as the enemy release hypothesis (Enders et al. 2020; Keane and Crawley 2002), we are far from fully comprehending what drives successful invasion. A promising approach to understanding the mechanisms that allow for invasion is to understand the factors that regulate species performance in the native range. For example, in their native range, invasive species might be those that take advantage of resource-rich environments by rapidly uptaking and depleting available resources, but then become limited by specialized enemies once they become abundant. In their secondary range, these species might be less limited by specialized enemies leading to possibly less negative intraspeci c plant-soil feedback ( Plant-soil feedback (PSF) is de ned as abiotic and biotic changes of soil by plants that subsequently alter plant growth (Bever et al. 1997). PSF has been suggested to play a role in plant invasions. Invasive species often create more positive (or less negative) PSF compared to native species (Chiuffo et al. 2015; Engelkes et al. 2008;Klironomos 2002; or non-invasive alien species (Aldorfova et al. 2020). Further, PSF has been shown to be more positive (or less negative) in the introduced compared to the native range of the plant species (Callaway et al. 2011; Reinhart and Callaway 2004; Reinhart et al. 2003). However, more studies in the native range are necessary to determine if plant performance of invasive plants is regulated by natural specialized enemies in their native range. Patterns by which plant species interact with the soil and soil biota in their native ranges might make some of them more prone to bene ting from pathogen release when introduced to the secondary range than others. In support of this hypothesis, Zuppinger-Dingley et al. (2011) showed on a set of 16 grassland species that species that are invasive in some region of the world show more negative PSF in their native ranges than their non-invading relatives.
There are many different taxa involved in the biotic component of PSF, including bacteria, arbuscular mycorrhizal fungi (AMF), non-mycorrhizal fungi, protozoans, nematodes and microarthropods, and these groups can all have positive, negative or net neutral effects on the plants. Bacteria and non-mycorrhizal fungi are primarily associated with negative PSF effects however, they may enhance plant performance as well, either via direct growth promotion in case of plant growth promoting rhizobacteria (Vacheron et al. 2013; van Loon 2007), or via increasing decomposition rates and nutrient availability (Weidner et al. 2015). On the contrary, AMF are usually considered to be bene cial for the plants, however, these may act as pathogens and reduce plant performance, especially in highly productive environments (Johnson et al. 1997). Soil mesofauna, comprising nematodes or various microarthropods, vary in their effects on plant performance, including negative effects of pathogens or root feeding organisms as well as positive effects of detritovores or organisms feeding on harmful microbiota (Bonkowski et al. 2009). Ideally, PSF experiments should identify the individual groups of soil biota that are involved in positive and negative interactions with plants ( Dawson and Schrama 2016). In order to assess how speci c soil biota affect plant performance, one would need to isolate the biota, prepare pure cultures and inoculate plants with them, which is extremely time consuming (Dawson and Schrama 2016). An easier, yet valuable approach is to take advantage of the fact that soil biota largely varies in size and inoculate the soil with whole or partial soil biota obtained by ltering soil solutions. By doing so, one can for example separate the effects Here, we quanti ed PSF-related mechanisms of invasion of Cirsium vulgare (Asteraceae), a species that is native to Europe, but has successfully naturalized on every continent except Antarctica, and is highly invasive in some areas (Julien and Gri ths 1998; Tenhumberg et al. 2008). We compared plant-soil interactions of C. vulgare (hereafter the invasive species) in its native range in Europe with plant-soil interactions of its native congener, C. oleraceum, that is not known to be naturalized or invasive anywhere in the world (hereafter referred to as the non-invasive species). Speci cally, we used structural equation modeling to understand how the abiotic and biotic pathways in PSF in uence plant performance. This requires quantifying how the species condition the soil, both in respect to changes in soil nutrient levels and composition of soil biota, how plant performance responds to soil conditioning, and which groups of soil biota (e.g., bacteria, fungi, AMF) are driving these changes in plant performance. Most studies address PSF solely in terms of aboveground biomass of the plants as it is the easiest measure of plant performance (Kardol et al. 2013). However, it has been shown that PSF can change in intensity and even in direction throughout plant's life (Aldorfova et al. 2020;Dudenhoffer et al. 2018;Florianova and Munzbergova 2018) and that PSF depends on, and thus probably also affects, the size of the root system and allocation to root biomass (Aldorfova and Munzbergova 2019; Bergmann et al. 2016;Cortois et al. 2016). We therefore used three measures of plant performance: seedling emergence, aboveground biomass, and root-shoot ratio.

Material And Methods
Studied species and seed collection For this study, we selected a pair of congeneric species, Cirsium vulgare (the invasive species) and Cirsium oleraceum (the non-invasive species), Asteraceae, Carduoideae. Both species are native to Europe, with one of their ecological optima being in ruderal vegetation. Speci cally in the Czech Republic, C. oleraceum is more frequent and abundant than C. vulgare [occurrence frequency in vegetation plots 4.1% vs 1%, mean percentage cover 7.8% vs. 2.3%, and maximum percentage cover 88% vs. 38% (Wild et al. 2019)]. However, globally, and especially in North America, C. vulgare is reported to be a noxious weed and highly invasive species (Julien and Gri ths 1998; Sieg et al. 2003;Tenhumberg et al. 2008), while C. oleraceum has never been reported as an invader elsewhere.
Seeds of both species were collected in the eld in the Czech Republic in 2017. For each species, we collected mature seeds from at least 10 individuals. Seeds from all individuals were mixed and mother plants were not further distinguished in the experiment. All collected seeds were surface sterilized with a diluted SAVO Originál (a 4.7% sodium chlorite-based disinfectant) prior to the experiment to reduce the chance of soil contamination via seed surface fungi.

Experimental design
Following a commonly used methodology (Bever et al. 1997;Kulmatiski et al. 2008), the plants were grown in a two-phase experiment. In the rst (conditioning) phase, conditioned soil was prepared. Soil biotic and abiotic characteristics were assessed after the conditioning phase to compare the effect of the two species on the soil. In the second (feedback) phase, we studied intraspeci c PSF by growing the plants in 12 different types of soil, including conditioned and unplanted control soil with full, partial or no soil biota.

Conditioning phase
The aim of the conditioning phase was to prepare the soil, conditioned by the species, for the upcoming feedback phase. The conditioning phase was carried out between April 2018 and July 2018 in the experimental garden of the Institute of Botany of the Czech Academy of Sciences (49°59′38.972′′N, 14°33′57.637′′E), 320 m above sea level, temperate climate zone, where the mean annual temperature is 8.6 °C and the mean annual precipitation is 610 mm.
To set up the conditioning phase, we used a mixture of topsoil (purchased from JENA company) and sand (AGRO Jesenice) in 1:1 ratio (for chemical characteristics of the soil mixture see Table S1). For each species, we used 150, 1-liter pots (10×10×10 cm) in the conditioning phase. Half of the pots were sown with 10 seeds of one of the species in April 2018, the other half of the pots remained unsown and served as controls. It is important to stress that even though the control pots remained unplanted during the conditioning phase, the soil was still a live soil in which a mixture of plant species was previously grown, and it thus contained species non-speci c soil biota. Each pot with conditioned soil was randomly assigned its unplanted control pot. The pairs of pots were always kept in close proximity to each other throughout the experiment so that they were exposed to exactly the same conditions. Both pots with and without plants were kept under the same conditions, regularly watered with tap water, and weeded on a weekly basis to avoid any effects of other species on the soil.
After the seeds germinated and the seedlings emerged, we randomly removed some of the seedlings to keep just one seedling per pot. The soil was conditioned for 12 weeks, similar to a range of previous studies (e.g., Chiuffo  After the harvest, the pots with each species as well as their paired unplanted control pots were randomly divided into ten groups of 7-8 pots and their soil was mixed. For each species we thus had 10 heaps of conditioned soil and 10 paired heaps of control soil. Heap served as a replicate and was further treated as such. From each heap, one sixth of the soil was collected for analysis of soil chemistry and soil biota, one third was kept untreated to serve as source of speci c biota for soil inoculation in the feedback phase, and the rest was sterilized by gamma irradiation (sterilization dose 25 kGy, performed by Bioster a.s. in Veverská Bítýška) and used as a background soil in the feedback phase.

Feedback phase
The feedback phase was carried out between September 2018 and March 2019 in a greenhouse of the Institute of Botany of the Czech Academy of Sciences. The greenhouse was heated to 18°C and daylight was extended by two hours every day.
In the feedback phase, we grew each species in six treatments of conditioned soil and six treatments of unplanted control soil. These treatments included sterilized soil, sterilized soil inoculated with microbial ltrate of conditioned or control soil, sterilized soil inoculated with whole inoculum of conditioned or control soil, and non-sterilized whole soil (Fig. 1). Inoculum and ltrate always originated from the same heap of soil as the background soil or from their paired heap with different soil conditioning. Using these treatments, we can assess the role of individual components in the PSF. By comparing growth in control and conditioned non-sterilized whole soil we can assess total net PSF. By comparing growth in control and conditioned sterilized soil, we can assess the effect of abiotic PSF [although there are complications with nutrient enrichment due to soil sterilization by gamma irradiation (McNamara et al. 2003;Troelstra et al. 2001), see Discussion for more details]. By comparing growth in sterilized soil with growth in soil with microbial ltrate we can quantify the effect of microbiota (bacteria and non-mycorrhizal fungi). By comparing growth in soil with microbial ltrate with growth in soil with whole inoculum the effect of other groups of soil biota can be assessed. By comparing growth in sterilized soil with growth in soil with whole inoculum total biotic PSF effects can be assessed. By comparing growth in soils with ltrates or inocula from conditioned and control soils, we can assess the effect of soil biota abundance and/or speci city, assuming conditioned soils have higher abundances of soil biota, as well as more speci c soil biota compared to control soils.
To set up the feedback phase, we used 10, 1-liter pots (10×10×10 cm) per species, soil conditioning and treatment, resulting in 120 pots per species, 240 pots in total. The bottoms of the pots were covered with keramzit sterilized in autoclave up to the height of 2 cm to compensate for soil lost during the harvest of the conditioning phase, and the rest of the pots was lled with 500 ml of soil mixed depending on the treatment. For non-sterilized whole soil treatments, we used untreated soil from the conditioning phase of the experiment. For whole inoculum treatments, we mixed sterilized soil and untreated soil from the conditioning phase in a 9:1 ratio. For the treatments with microbial ltrate, we lled the pots with sterilized soil and we watered them with the microbial ltrate. The ltrate was created by mixing 50 ml of untreated soil in 500 ml of distilled water, homogenizing the mixture, and ltering it through two lter papers with pore size of 11 μm. Therefore, the microbial ltrate does not contain micro-arthropods, nematodes, or arbuscular mycorrhizal fungi, whereas it should contain soil bacteria and fungi (van de Voorde et al. 2012). For sterilized treatments, we lled the pots with sterilized soil and we watered them with microbial ltrate sterilized in autoclave.
Each pot was sown with 9 seeds of the same species as in the conditioning phase. The pots were kept in the greenhouse, regularly watered, and weeded when needed. All pots originating from one pair of heaps were kept in the same block within the greenhouse. Seedling emergence was followed. Three weeks after the rst seedlings emerged in all pots, all seedlings but one were removed from each pot to avoid competition. All seedlings emerging afterwards were recorded and removed as well. Twelve weeks after germination, the plants were harvested, divided into above-and below-ground biomass and weighed. All pots of both species were harvested at the same time.

Soil characteristics
Soil characteristics were analyzed after the conditioning phase for three types of soil: soil conditioned by the invasive species, soil conditioned by the non-invasive species, and the control soil. For each of the soil conditioning types, samples from six out of the ten heaps were randomly selected for the analyses. In addition, the analyses were performed also on soil collected before the conditioning phase (Table S1).
From abiotic soil characteristics, we measured actual and exchangeable pH, total C, N, P and available P, Ca, Mg, and K. From biotic characteristics, we determined soil microbial community composition using phospholipid and neutral fatty acids analysis (PLFA and NLFA) and we assessed the infection potential Abiotic soil characteristics Actual and exchangeable pH was measured using deionized water and 0.1M solution of KCl as extracting solutions, respectively (ISO 10390: Soil quality -Determination of pH. International Organization for Standardization, ISO 2000). Total C and N contents were determined by methods of Ehrenberger and Gorbach (1973) using CHN catalyst (Carlo Erba NC 2500), total P was measured according to the method of Olsen and Sommers (1982). Available P was measured in ltrate of 5 g of soil with 50 ml of 0.5M K 2 SO 4 solution by ow injection analysis with spectrophotometric detection using the instrument QuikChem FIA + 8000 Series (Ammerman 2001;Egan 2001). Concentrations of available Ca 2+ and K + were measured using atomic emission spectrometry method and available Mg 2+ using atomic absorption spectrometry according to methods of Moore and Chapman (1986) and Dědina (1987), with solution of 1M ammonium acetate as the extractant. All analyses were performed by the Analytical Laboratory of To assess MPN and MIP, we evaluated mycorrhizal colonization of maize roots [standardly used for assessing MPN and MIP as its roots are strongly colonized by AMF (Moorman and Reeves 1979)] that was grown in each of the studied types of soil in 1:0, 1:10, 1:100, 1:1000, 1:10000 dilutions with soil sterilized in autoclave, in ve replicates per dilution. Maize seeds (Zea mays convar. saccharata, var. Ashworth) were purchased from a commercial supplier (ReinSaat KG company, St. Leonhard am Hornerwald, Austria), they were germinated in Petri dishes in sterile conditions, replanted into 100 ml plastic containers (4×14 cm), and left growing in a greenhouse. After six weeks, the plants were harvested, ne roots from the middle part of the root system were collected, placed in 10% KOH for three months to bleach, and stained (left for 12h in 2% lactic acid, 12h in 0.05% trypan blue in lactoglycerol, rinsed in water, and soaked into lactoglycerol prepared from glycerol, 80% lactic acid and distilled water in 3:2:5 ratio).
The stained roots from 1:10, 1:100, 1:1000, and 1:10000 dilutions were observed using a binocular magni er and presence of AMF propagules was recorded. MPN/ml was calculated using a program MPN Calculator, Build 23 using information on types of dilutions, number of replicates per dilution and number of replicates per dilution in which AMF propagules were recorded. To assess MIP, only the 1:10 dilution was used. Stained roots were placed into a Petri dish with a 1x1 cm grid and presence of AMF propagules at 200 intersections of roots with the grid was recorded using a binocular magni er. An average value from the ve replicates was calculated both for MPN and MIP, resulting in one MPN and one MIP value per soil sample and six replicates per soil conditioning type.

Statistical analyses
Differences in soil biotic and abiotic characteristics between soil conditioned by C. arvense, and by C. oleraceum were studied using linear direct gradient analysis (Redundancy Analysis, RDA) and Monte-Carlo permutation tests (Ter Braak and Šmilauer 2012) with 499 permutations. Dependent variables used in this analysis were all the studied soil characteristics except for actual pH, K content, total microbial and bacterial biomass, which were excluded due to high correlations with other variables (Table S2). The variables were standardized prior to the analysis. The independent variable was conditioning species. We repeated the analysis with all three soil conditioning types including the control soil and we present the results in the appendix (Fig. S1). The analyses were performed in Canoco 5 (Ter Braak and Šmilauer 2012). As a supplementary analysis, we also performed ANOVA using R 3.6.1 (R Core Team 2019) always with one of the studied soil characteristics as dependent variable and tested the differences between multiple levels of soil conditioning type using Tukey post hoc tests (Fig. S2).
Differences in plant performance between individual treatments and soil conditioning types in the feedback phase were tested using a linear (square root transformed biomass and root-shoot ratio) or generalized linear (seedling emergence as number of emerged seedlings out of the number of seeds sown, with binomial error distribution) mixed effect models in the R package 'lmerTest' (Kuznetsova et al. 2017) with identity of the soil heap as random effect, and species, soil conditioning, treatment (sterilized soil, ltrate from control soil, ltrate from conditioned soil, inoculum from control soil, inoculum from conditioned soil, non-sterilized whole soil), and their interactions as explanatory variables. To estimate pvalues, we used F-tests comparing two models with and without a tested term, using a 'drop1' function in the 'lmerTest' package (Kuznetsova et al. 2017). To assess differences between pairs of group means, we used Tukey post-hoc tests adapted to mixed effect models using 'glht' function in 'multcomp' Rpackage (Hothorn et al. 2008).
Afterwards, we used subset of data excluding the sterilized treatment and the non-sterilized whole soil treatment and tested the effect of the type of soil biota ( ltrate vs inoculum) and conditioning of soil biota (from control vs from conditioned soil) as explanatory variables instead of the treatment variable, otherwise there were no changes in the analyses. We obtained very similar results when including only the subset of treatments in the analyses and so we only present these results in the Results section. Results of the analyses including all the treatments and not differentiating between type and conditioning of soil biota can be found in the appendix (Table S3). The two treatments which are excluded from the main analyses are, however, visualized in some of the graphs and compared using multiple comparisons with the rest.
Last, we used structural equation modeling [performed in the 'lavaan' package (Rosseel 2012) in R] to assess how individual components of soil, i.e. amount of soil nutrients, bacterial, fungal and AMF biomass, affect biomass of the two species. For the analysis, we only used data on plant biomass from the non-sterilized whole soil treatment as detailed soil analyses are only available for this treatment. A separate model was created for each species. The assumed relationships were as follows: (i) plant performance is affected by the amount of soil nutrients and by bacterial, fungal and AMF biomass, (ii) bacterial, fungal and AMF biomass are affected by the amount of soil nutrients, and (iii) bacterial, fungal and AMF biomass are correlated.

Results
Effect of conditioning species on soil characteristics Soil characteristics signi cantly differed between soils conditioned by the invasive and the non-invasive species (Pseudo-F = 6.0, p = 0.004, 37.59% of explained variation, Fig. 2). Values of MPN and AMF were higher in soils conditioned by the invasive species (Fig. 2, Fig. S2), and in both cases the values were much higher than in the control soil (Fig. S1, S2). Nutrient levels and both bacterial and fungal biomass were higher in soils conditioned by the non-invasive species (Fig. 2, Fig. S2).

Effect of soil conditioning and treatments on plant performance
The invasive species had signi cantly higher seedling emergence and lower root-shoot ratio than the noninvasive species (Table 1, Fig. S3). The two species did not differ in total biomass production ( Table 1).
Soil conditioning negatively affected plant biomass, with no differences between the two species ( Table  1, Fig. S4). Type of soil biota had a signi cant effect on plant biomass and root-shoot ratio ( Table 1). In both cases, the values were the highest in microbial ltrate treatments, lower in whole soil inoculum and the lowest in whole soil (Fig. S5). The effects did not differ between the two species ( Table 1).
Effect of conditioning of soil biota, i.e. whether the biota originated from control or conditioned soil, on biomass and root-shoot ratio differed between the two species (Table 1). In both cases, the non-invasive species bene ted from growth with conditioned biota compared to biota from control soil, while the invasive species performed similarly with both conditioned and control biota (Fig. 3).
The interaction between species, type of soil biota and soil biota conditioning was not signi cant for any of the measures of plant performance (Table 1). However, the interaction of species and treatment (comprising type and conditioning of biota) was signi cant for seedling emergence in the analysis using all treatments (Table S3). For the invasive species, seedling emergence in presence of microbial ltrate from control soil was higher than in microbial ltrate from conditioned soils as well as the sterilized soil, but no differences in seedling emergence among the treatments were found for the non-invasive species (Fig. 4).
The interaction between species, soil conditioning, type of soil biota, and conditioning of soil biota was signi cant for biomass and root-shoot ratio (Table 1). For the non-invasive species, conditioning of microbial ltrate had a positive effect on biomass in conditioned but not in control soil, while for the invasive species it had a positive effect in control but not conditioned soil (Fig. 5a). Root-shoot ratio was negatively affected by conditioning of soil inoculum in both control and conditioned soil for the invasive species, but for the non-invasive species only in control, not in conditioned soil (Fig. 5b).

Determinants of plant performance
Structural equation models showed that the determinants of plant performance in the non-sterilized whole soil treatment differ between the non-invasive and the invasive species. The non-invasive species responded negatively to bacterial biomass and positively to fungal biomass and soil nutrients levels, while the invasive species responded positively to bacterial biomass and was not signi cantly affected by fungal biomass or soil nutrients. While both species responded negatively to AMF biomass, only the invasive species signi cantly increased the AMF biomass as it depleted soil nutrients.

Discussion
In the present study, we found differences in plant-soil interactions between Cirsium vulgare and C. oleraceum, both native to Europe but only C. vulgare invasive elsewhere, which suggest a possible role of these interactions in the invasive potential of C. vulgare. Compared to its non-invasive congener, the invasive C. vulgare more rapidly depleted nutrients from the soil, was less in uenced directly by the availability of soil nutrients, and had more negative biotic PSF, particularly PSF caused by larger-sized soil biota. The invasive species also had higher seedling emergence and lower root-shoot ratio compared to non-invasive species and showed greater ability to decrease its root-shoot ratio in the presence of harmful soil biota. These results highlight that experimental studies from the native range on PSF can offer important insight in our understanding of processes that regulate invasive plant populations.
Soils conditioned by the invasive species had lower levels of nutrients, particularly of P, N and Ca (Fig. 2). This is in line with previous research showing invasive species often exploit soil nutrients more e ciently than non-invasive species (Dassonville et al. 2008;Funk and Vitousek 2007;Sardans et al. 2017), allowing them to gain competitive advantage over other species. Importantly, despite lower nutrient levels in soils conditioned by the invasive species, this species did not show more negative abiotic PSF than the non-invasive species, but, on the contrary, responded to soil conditioning in sterilized soil slightly less negatively than the non-invasive species (Fig. 5a). In line with that, the structural equation model showed lower sensitivity of the invasive species to soil nutrients than the non-invasive species (Fig. 6). This means that the invasive species copes better with altered nutrient levels, indicating its higher plasticity in response to nutrients, a common feature of successful invaders (Burns 2004;Daehler 2003;Funk 2008).
Both plant species had lower biomass with increasing amounts of soil biota (Fig S5), showing that the negative effects of soil biota prevail over the positive effects, both for microbiota and larger-sized soil biota, as has been shown in previous research (van de Voorde et al. 2012;Wang et al. 2019b). The invasive species had more negative biotic PSF, particularly PSF driven by the larger-sized biota, than the non-invasive species (Fig. 3, Fig. 5) when de ning PSF as a difference in performance of plants grown with biota from self-conditioned ('home') and control ('away') soil, as recommended when the research question concerns the speci city of soil feedback effects (Brinkman et al. 2010;Cortois et al. 2016). It is unclear in our study if this result is due to the differences in composition of the self-conditioned biota (i.e., more speci c enemies) or just their abundance since both are likely to differ from an unplanted control. Bacterial biomass had an overall negative effect on the biomass of the non-invasive species, but positive on the invasive species (Fig. 6), suggesting that the invasive species was less affected by bacterial pathogens and bene ted more from bacterial mutualists. There is a large chance that the species bene ts from presence of bacterial mutualists in the secondary range as well since most mutualists are quite generalistic (Bronstein 2003) and invasive plants can often form mutualisms as effective or even more effective in the new ranges than in the old range (Parker and Gilbert 2007;Richardson et al. 2000). Interestingly, both species bene ted from growth with their self-conditioned microbiota compared to the control microbiota and this was more pronounced for the non-invasive species (Fig. 5a). This result may partially explain the differences in success of the two species when introduced into a secondary range. When moving to secondary range, the plants are supposed to leave their specialized soil biota behind and only be affected by the local generalist biota, which in case of the invasive C. vulgare seem to have more positive effects on its performance.
In contrast, the invasive species responded much more negatively to self-conditioned larger-sized soil biota compared to the control biota (Fig. 5), providing more opportunities for the invasive species to bene t from enemy release when introduced to the secondary range than for the non-invasive species.
Larger-sized biota in our study refers to mesofauna such as nematodes and microarthropods, but also AMF. Since we only quanti ed AMF but did not study composition of the mesofauna, we cannot say which groups contributed the most to the negative PSF and the differences between species. AMF had a net harmful effect on both plant species. In the literature, more studies nd net positive effects of AMF on plants, however, there are several other studies that report negative effects of AMF (Janos 2007;Johnson et al. 1997), particularly in nutrient-rich soils such as the ones used in our experiment. Only the invasive plant had a PSF in which it increased the biomass of harmful AMF when it depleted soil nutrients (Fig. 6). Even though AMF have relatively low host speci city, host preference in natural ecosystems has been identi ed (Sanders 2003). Because AMF composition differs between world regions (Sturmer et al. 2018), chances are that the invasive species leaves behind some of the harmful AMF when moving to the secondary range, which would further contribute to its invasion success. However, since we do not have data on AMF species composition or on the effect of AMF on the species in its secondary range, this explanation remains purely hypothetical.
Root-shoot ratio of both species decreased when more soil biota was included (Fig. S5). Since the size of , reducing allocation into root biomass in presence of detrimental soil biota may serve as a protective mechanism for the plants, minimizing the negative effects of soil biota on plant growth. Besides the response to the type of soil biota included, the invasive species also decreased its root-shoot ratio in presence of self-conditioned biota compared to control biota in case of the larger-size biota (Fig. 5b), pointing to its greater sensitivity to specialized biota and possibly greater plasticity in biomass allocation than the non-invasive species.
Seedling emergence was overall higher for the invasive species and did not differ between whole soil inoculum treatment and sterilized soil for either species. Microbial ltrate from control, but not from selfconditioned soil, however, increased seedling emergence of the invasive species. These results suggest that non-specialized microbes, i.e. those the species is relatively more likely to encounter in the secondary range, bene t plant performance of the invasive species in early stages of their life. This, combined with the high overall seedling emergence, may be another factor contributing to the invasiveness of C. vulgare. Despite the recent recognition that plant-soil interactions shape seedling performance and that these interactions are often positive (Aldorfova et al. 2020;Dudenhoffer et al. 2018;Florianova and Munzbergova 2018), most PSF research focuses solely on PSF of adult plants (Kardol et al. 2013) and the mechanisms involved in PSF of juveniles remain largely unexplored. The positive PSF effects have usually been attributed to AMF, as seedlings are expected to bene t more from associations with AMF than adult plants due to a less developed root system (Aldrich-Wolfe 2007; van der Heijden 2004). Our study in contrast nds an important role of soil microbiota rather than of AMF. It is not clear which speci c group of soil microbiota was responsible for the effects. It might have been plant growthpromoting rhizobacteria which positively affect seed germination (Kloepper et al. 1991;Wu et al. 2016) and which may be stimulated by root exudates released by some plants (Hu et al. 2018;Vacheron et al. 2013; van Loon 2007), but more investigation is needed to understand the precise underlying mechanisms of these interactions.

Conclusions
We showed that plant-soil interactions of invasive Cirsium vulgare in its native range may help to explain its invasive success elsewhere, by comparing it with plant-soil interactions of C. oleraceum which is not known to be invasive anywhere else in the world. This invasive species is able to reduce nutrients to lower levels but maintain its high performance regardless of soil nutrient levels. While soil bacteria in general have more positive effect on the invasive species, the invasive species bene ts less from growth with specialized (self-conditioned) microbiota compared to generalist (control) microbiota. On the other hand, it is relatively more harmed by self-speci c larger-sized soil biota. Since the specialized biota is less likely to be present in the secondary range than the generalist biota, our results suggest that the invasive species may bene t more from pathogen release and at the same time suffer less from loss of specialized mutualists when transferred to the secondary range than the non-invasive species.

Declarations
Funding -This study was supported by The Czech Science Foundation (project GAČR 20-01813S), and partly also by institutional funding (RVO 67985939 and the Ministry of Education of the Czech Republic).   Effect of treatment (type and origin of soil biota) on seedling emergence for individual species. Bars and error lines represent mean ± SE. Bars that share the same letter do not signi cantly (p < 0.05) differ from each other after Tukey post-hoc tests