Don’t be naïve: Eco-evolutionary experience better explains invasion success of Senecio inaequidens than soil conditions

20 Introduced species encounter novel biotic and abiotic conditions that influence their 21 success in new environments. Their advantage is often linked to reduced competition 22 from native species that lack eco-evolutionary experience, as well as to their ability to 23 pre-empt resources. Once established, their success can also be shaped by changes in 24 soil conditions, particularly through interactions with soil microbial communities. 25 Understanding how these factors influence invasion success can provide valuable 26 insights into predicting future invasions under global change. In this study, we examined 27 how eco-evolutionary experience and soil bacterial communities influenced the 28 performance of the invasive subshrub Senecio inaequidens DC. We conducted a fully 29 factorial experiment in growth chambers consisting of two factors: competing community 30 identity with three levels (plant species from its native range (South Africa), from its 31 invasive range (Italy) and a control with only S. inaequidens) and soil biota conditions 32 with two levels (wild soil and autoclaved soil with lower microbial load). Our results 33 showed that plant community identity had the strongest effect on S. inaequidens growth 34 (height and lateral spread), with the smallest individuals occurring in competition with 35 South African species. Growing on autoclaved soil had no major impact on plant height, 36 suggesting that reduced competition played a greater role than soil bacterial differences 37 in determining plant performance. Suppression was stronger when the competing native 38 species were more closely related to S. inaequidens. Soil bacterial communities were 39 influenced by both plant identity and soil treatment, and S. inaequidens performed 40 better in soils with lower bacterial diversity, possibly due to reduced pathogen pressure. 41 These findings suggest that invasive species management could be improved by 42 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 3 fostering competition with evolutionarily experienced native species and maintaining or 43 enhancing soil microbial diversity to limit invader success. 44

eco-evolutionary experience; phylogenetic similarity, plant traits; relatedness; 45 soil bacteria; South African ragwort 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 4

60 Upon entering new environments, introduced species encounter novel biotic and abiotic 61 conditions that can either facilitate or hinder the invasion process (Heger et al. 2019). 62 To succeed, these species must effectively establish new interactions, make use of and 63 compete for available resources to eventually become invasive (Funk and Vitousek 64 2007). For example, introduced plants may encounter new competitive, pathogenic or 65 herbivore pressures (Funk et al. 2008), lose critical mutualistic interactions (Mitchell et 66 al. 2006) or face novel traits such as allelochemical release, to which they lack eco-67 evolutionary experience (Callaway and Ridenour 2004). Understanding the mechanisms 68 underlying the success of species introduced in new areas can provide valuable insights 69 into predicting other processes, such as the expansion of species ranges and potential 70 future invasions in the context of global change (Fristoe et al. 2021). 71 The outcome of novel interactions between introduced and native species may be 72 shaped by their evolutionary history, particularly their past experiences interacting with 73 specific species or traits (Saul et al. 2013; Saul and Jeschke 2015). In this context, the 74 invasion success of introduced plants might be hindered if the recipient community 75 includes closely related species, as these species or other community members are 76 more likely to have eco-evolutionary experience with similar competitors, predators, or 77 other antagonistic interactions (Saul et al. 2013). Conversely, functionally dissimilar 78 introduced species with novel physiological and morphological traits, such as the ability 79 of exudating allelopathic compounds or the ability to exploit untapped resources, may 80 gain a competitive advantage in communities where native species lack eco-81 evolutionary experience with these traits. This experience can influence how native 82 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 5 species respond to novel competitors, thereby shaping invasion dynamics (Heger and 83 Trepl 2003; Heger et al. 2019; Novoa et al. 2020). 84 At the community level, niche overlap and resource preemption due to competition for 85 limited resources, are key factors in the success of introduced plant species 86 (MacDougall et al. 2009). Darwin's naturalization hypothesis posits that introduced 87 species would struggle to establish in communities with closely related native species 88 but have a higher invasion success when they are more phylogenetically distant from 89 the resident flora (Darwin 1859; Daehler 2001; Yannelli et al. 2025). The underlying 90 assumption is that niche similarity among species is phylogenetically conserved 91 (Prinzing 2001), with phylogenetic relatedness reflecting shared traits that influence 92 species ability to coexist (Blomberg and Garland 2002). This success is further shaped 93 by the species competitive ability; both in terms of its competitive effect, or its capacity 94 to suppress other individuals by depleting resources, and its competitive response, or its 95 ability to tolerate growth suppression from neighboring plants (Goldberg 1990). While 96 the hypothesis has shed light on initial establishment (Park and Potter 2013; Yannelli et 97 al. 2017), inconsistencies arise from temporal variations and shifts in traits among 98 closely related species (Burns and Winn 2006; Thuiller et al. 2010; Li et al. 2015). To 99 better understand the drivers of invasion success, it is crucial to test this hypothesis with 100 species from both native and invasive ranges, a perspective that has yet to be fully 101 explored (but see e.g. Zheng et al. 2018). 102 Resource availability and competition can be influenced by belowground dynamics 103 linked to plant traits and microbial communities. Soil organisms play a crucial role in 104 mediating interactions between native and invasive plant species, affecting e.g., 105 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 6 competitive, mutualistic, and pathogenic interactions, and ultimately invasion success 106 (Abbott et al. 2015; Fahey and Flory 2022). The competitive strength of invasive plants 107 may be increased by altered microbial communities if they lose coevolved specialist 108 pathogens from their native range (Mitchell and Power 2003; Fahey and Flory 2022) or 109 serve as reservoirs for pathogens that disproportionately affect native species (Eppinga 110 et al. 2006; Mangla and Callaway 2008). New interactions with pathogenic or mutualistic 111 microorganisms can be established with invasive species in the new area based on 112 similarities in traits to those of native species, even without any previous evolutionary 113 experience (Eppinga et al. 2006; Diez et al. 2010). Trait differences in invasive plants 114 compared to the recipient communities, particularly those associated with acquisitive 115 strategies, can also lead to shifts in the microbial community from fungi- to bacteria-116 dominated (Ehrenfeld 2003; Wardle et al. 2004; Torres et al. 2021). Nevertheless, the 117 role of soil bacteria in mediating competition and plant community assembly, in 118 particular through pathogenic interactions, seems highly limited (van der Putten et al. 119 2007; Dawson and Schrama 2016). 120 We selected the invasive plant Senecio inaequidens DC., commonly known as “South 121 African ragwort” or “Canary Weed”, as our study species (hereafter sometimes referred 122 to just with its generic name). This perennial chamaephyte native to South Africa, and 123 invasive in areas of the country outside of its native range, was introduced to Europe in 124 the late 19th century (Ernst 1998) and has become invasive in disturbed areas. Senecio 125 inaequidens has been found to produce allelopathic defenses in the form of pyrrolizidine 126 alkaloids (Joosten and van Veen 2011), which can protect it against both above- and 127 belowground herbivory (Caño et al. 2009; Thoden et al. 2009), influencing soil microbial 128 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 7 communities (Harkes et al. 2017). Senecio inaequidens traits were also found to 129 correlate with rhizosphere biota, with bacterial diversity being positively associated with 130 resource allocation to belowground growth and late flowering (Thébault et al. 2010). 131 Furthermore, Van De Walle et al. (2022) found S. inaequidens to modify soil abiotic 132 conditions, increasing nutrient concentrations via litter deposition and eliciting increased 133 growth of co-occurring native species in nutrient-poor habitats. However, the impact of 134 these traits and soil community alterations on its competitive success in the invasive 135 versus native range has remained unexplored. 136 In this study, we investigated S. inaequidens competitive response to native plant 137 species from both its native and invasive ranges under controlled experimental 138 conditions. The experimental communities included species with which S. inaequidens 139 shares a history of eco-evolutionary interactions (native range) and species to which it is 140 evolutionarily naïve (invasive range), lacking such historical associations. We also 141 examined how soil conditions, including the presence or absence (by autoclaving) of 142 soil biota from the invasive range, influenced these competitive interactions. 143 Specifically, we explored whether eco-evolutionary experience, phylogenetic 144 relatedness, and soil biota could explain Senecio's performance when competing with 145 native plant communities. We hypothesized that: (i) S. inaequidens performance will 146 depend on the identity of the competing plant communities, and it will perform better 147 when competing with naïve species from the invasive range compared to experienced 148 species from the native range; (ii) an increase in phylogenetic relatedness between S. 149 inaequidens and the competing species in the community will result in lower 150 performance of S. inaequidens, following Darwin’s naturalization hypothesis; and (iii) 151 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 8 soil biota will influence the competitive interactions between S. inaequidens and native 152 plant communities, with reduced biotic effects in autoclaved soils leading to better 153 performance of S. inaequidens compared to non-autoclaved soils. 154

155 Plant and soil material collection 156 Senecio inaequidens is a subshrub that belongs to the Asteraceae family, often 157 reaching 40-100 cm in height. Native to South Africa's highlands, it was introduced to 158 Europe as a wool contaminant (Ernst 1998). It thrives in disturbed areas like roadsides, 159 railways, and quarries, as well as dry grasslands, pastures, and vineyards (Heger and 160 Böhmer 2005; López-García and Maillet 2005). In its native range, the species exists in 161 diploid (2n = 20) and tetraploid (2n = 40) forms, but only tetraploids are found in Europe 162 (Lafuma et al. 2003). 163 Seeds of S. inaequidens were collected from a population located in the former quarry 164 of Collepedrino, Northern Italy, which is currently heavily invaded by this species 165 (Bergamo, 45°46'37.4"N 9°31'09.5"). To design the competing native communities, we 166 chose five species known to co-occur with S. inaequidens in each range (i.e., native and 167 invasive). To assess this in the native range of the invasive species (i.e., South Africa), 168 we used the National Collections database (http://posa.sanbi.org/) to select species 169 documented in the area where tetraploid populations of S. inaequidens have been 170 reported (Lafuma et al. 2003). Upon availability in local seed companies, we refined the 171 list of native species by a second check against the results of vegetation surveys (Du 172 Preez and Bredenkamp 1991), to be sure that all natives would have co-occurred in a 173 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 9 plot-sized area. As a result, we selected a multi-species suite comprising Aristida 174 congesta Roem. & Schult. (Poaceae), Hibiscus trionum L. (Malvaceae), Salvia disermas 175 L. (Lamiaceae), Wahlenbergia androsacea A. DC. and Wahlenbergia undulata (L.f.) A. 176 DC. (Campunulaceae) for the native range. Seed material for the native range was 177 purchased at the local seed company Silverhill (Cape Town, South Africa). In the 178 invasive range (i.e. Italy), we selected native species according to known co-occurrence 179 in the Collepedrino quarry (Gentili et al. 2020) and collected seeds in the same area. 180 The list included Bromopsis erecta (Huds.) Fourr. (Poaceae), Hypericum perforatum L. 181 s.l. (Hypericaceae), Onobrychis viciifolia Scop. (Fabaceae), Poterium sanguisorba L. s.l. 182 (Rosaceae) and Trifolium repens L. (Fabaceae). We performed germination tests for all 183 native species to find the best conditions for their germination (Supplementary 184 Information, Section S1, Tab. S1). 185 Soil used for the experiment was collected in the same quarry. It was placed in open dry 186 bags and stored at room temperature until setting the experiment. We prepared the 187 experimental substrate by mixing the quarry soil, which was highly rocky, with common 188 potting substrate (TERCOM potting soil) in a 1:1 ratio to favor plant growth under 189 controlled conditions (growth chamber). Before setting the pot experiment, we 190 autoclaved half of this soil mix at 120°C for 45 minutes. 191 Experimental design and setting 192 Our experiment consisted of a fully factorial design with a combination of two factors: 193 competing community and soil biota. The competitive communities’ identity had three 194 levels, namely species from the native range considered to be experienced (SA; South 195 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 10 Africa), species from the invasive range considered to be naïve (IT; Italy) and the 196 control (CTR) with S. inaequidens individuals growing alone. Soil biota conditions had 197 two levels, i.e. autoclaved (st) and non-autoclaved soil (w; henceforth “wild”). Each 198 treatment combination was replicated five times, making up a total of 30 experimental 199 pots. Before the experiment, all seeds were stratified by placing them in paper bags at 200 4°C for about 1 month. Two different methods of germination were used to ensure the 201 survival of the seeds and high germination rates, according to results of germination 202 tests performed before the experiment: (1) Directly in plastic cups with a mix of 203 autoclaved common potting soil and sand with a 1:1 ratio; (2) in Petri dishes with 204 moistened filter paper, which were transplanted to plastic cups filled with autoclaved 205 common potting soil and sand at a 1:1 ratio, a few days after germination (see protocols 206 in Supplementary information, Section S1). When the seedlings were about 20 days old, 207 we placed two individuals of the invasive S. inaequidens in the middle of 2L pots filled 208 with a mix of quarry soil and common potting soil. At the same time, in all treatment 209 combinations that required competition with natives, we added 5 individuals for each 210 native species distributed at the edges of the pot. We then completely randomized the 211 pots and placed them in a growth chamber with an average temperature of 29°C, 212 relative humidity of 42%, and a day-night cycle of 14 and 10 hours, respectively. These 213 values were consistent with the growth conditions of S. inaequidens when invading 214 ruderal dry habitats (railways, roadsides, etc.). Plants were watered every other day for 215 the first days, and twice a week for the rest of the experiment. The experiment ran for 216 84 days, when some S. inaequidens individuals started to die. 217 218 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 11 Measurements and data processing 219 After 84 days from the start of the experiment, we collected data on plant vegetative 220 fitness and survivorship, as a proxy for success. Specifically, we measured the 221 maximum height as the shortest distance between the upper boundary of the main 222 photosynthetic tissues on a plant and the ground level, and the lateral growth of each 223 individual of S. inaequidens as the maximum width of the canopy (Pérez-Harguindeguy 224 et al. 2013). At this point, we also recorded the number and identity of the native 225 species that survived in each pot. 226 To assess the effect of relatedness on S. inaequidens performance, we calculated the 227 phylogenetic distances among all species in our experiment from a phylogenetic tree for 228 angiosperms as a backbone (Zanne et al. 2014) that was pruned from all species that 229 were not included in our experiment (Supplementary information, Fig. S1). We then 230 calculated community-weighted phylogenetic distances to the invader (CWMPD) by 231 weighting the native community-invasive distances with the proportion (based on the 232 number of individuals alive) of each species (in terms of number of individuals) in the 233 pot. Further, we also obtained the distance of the most abundant species in each 234 community to the invader (DMANS) to examine the effect of these species on S. 235 inaequidens growth. In the case of more than one species dominating the community in 236 the same abundance, we used total mean phylogenetic distances to every dominant 237 native. To characterize the soil bacterial communities in each treatment combination, 238 we collected soil samples at the end of the experiment (after 84 days) from three 239 randomly selected pots (n = 18). The samples were stored at -20°C until processing. 240 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 12 DNA extraction and Next Generation sequencing 241 Genomic DNA was extracted using the FastDNA® Spin Kit for Soil (MP Biomedicals, 242 Solon, OH, USA) following the manufacturer’s instructions. A first PCR amplification 243 was carried out using the 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 519R (5’-244 GWATTACCGCGGCKGCTG-3’) primers (Frank et al. 2008; Hollister et al. 2011) on the 245 original DNA extract and on the 1:10, 1:100, 1:1000 and 1:10000 dilutions, to detect the 246 possible presence of PCR inhibitors. Amplification conditions were: initial denaturation 247 at 95°C for 4 min, 29 cycles at 95°C for 30 s, 55°C for 45 s and 72°C for 45 s, and a 248 final extension at 72°C for 5 min. A second PCR was then performed using 783F and 249 1046R primers on the V5-V6 hypervariable regions of the bacterial 16S rRNA gene, with 250 customized oligonucleotide barcodes (6bp, see sequence in Table S2) added to their 5’ 251 end (Gandolfi et al. 2024). We used GoTaq® Green Master Mix (Promega Corporation, 252 Madison, WI, USA) and 1 µM of each primer, for a final volume of 2 x 50 µL for each 253 sample. This second amplification was performed under the following conditions: initial 254 denaturation at 94°C for 4 min, 28 cycles at 94°C for 50 s, 47°C for 30 s and 72°C for 255 30 s, and a final extension at 72°C for 5 min. The PCR products were purified using the 256 Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, 257 USA), following the manufacturer's instructions, and the DNA content was quantified 258 with the Qubit ® 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). Amplicon 259 libraries were prepared with nine samples each, identifiable due to different barcode 260 pairs. Library preparation with the addition of standard Nextera indices (Illumina, Inc., 261 San Diego, CA, USA) and sequencing with the MiSeq Illumina platform (Illumina, Inc., 262 San Diego, CA, USA), using a 2 × 250 bp paired-end protocol, was performed at the 263 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 13 Consorzio per il Centro di Biomedicina Molecolare (CBM), located in Trieste, Italy. 264 Amplicon Sequence Variants (ASVs) were inferred through the DADA2 algorithm 265 (Callahan et al. 2016), as described in Gandolfi et al. (2024). 266 Data analysis 267 All statistical analyses were performed using R version 4.3.1 (R-Core-Team 2023) and 268 the vegan package (Oksanen et al. 2022), unless stated otherwise. We used two-way 269 ANOVA to assess if the average height and lateral growth of S. inaequidens were 270 affected by the competing community, soil conditions and their interaction. Since there 271 was an imbalance in the experimental replication due to the mortality of S. inaequidens 272 in some replicates, we used the Type III test. We then performed post-hoc pairwise 273 comparisons with Tukey tests. In the same way, we used one-way ANOVA to test the 274 effect of native species identity on the height of S. inaequidens to explore the impact of 275 the presence of individual species. To evaluate the effect of CWMPD and DMANS in 276 each community on average S. inaequidens height and lateral growth, we used linear 277 regressions. 278 We used Non-metric Multidimensional Scaling (NMDS) analysis based on Bray-Curtis 279 dissimilarity distances (Bray and Curtis 1957) to visualize differences in soil bacterial 280 community structure according to the treatments using the metaMDS function. We 281 carried out a PERMANOVA test using the adonis2 function to assess treatment 282 combination effects on soil bacterial communities. Before performing these multivariate 283 analyses, we transformed the bacterial ASV abundance matrix with Hellinger distance 284 to reduce the emphasis on ASV abundances while highlighting their presence or 285 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 14 absence and mitigate the double-zero issue when comparing ASV compositions across 286 samples (Bocard et al. 2018). We calculated ASV richness and Shannon index for each 287 treatment combination on the rarefied bacterial data, which were based on the sample 288 with the lowest reading depth (2293). We then evaluated the effects of our treatments 289 on ASV richness using generalized linear models with a quasi-Poisson distribution to 290 correct for overdispersion present in the data (Cameron and Trivedi 1990) and used 291 ANOVA for the Shannon index. Finally, we explored the effect of bacterial alpha-292 diversity, i.e., ASV richness and Shannon index, on the height and lateral growth of S. 293 inaequidens using a linear model. 294

295 Effect of competition and soil biota conditions on Senecio performance 296 Senecio inaequidens performance was affected by the community it was growing along 297 with more than soil conditions, compared to the control treatment in pots where it grew 298 without competition. Specifically, in terms of S. inaequidens maximum height at day 84, 299 only community identity had a significant effect (ANOVA: Community: F = 4.31, p 0.05; Fig. 1). 302 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 15 303 Figure 1. Differences among community and soil treatments in height (left panel) and 304 lateral growth (right panel) of Senecio inaequidens 84 days after the experiment started. 305 CTR represents the control treatment with no native species growing with Senecio, IT is 306 the naïve community from the invasive range in Italy, and SA is the experienced 307 community from the native range in South Africa (ANOVA: Community F = 4.31, p < 308 0.03). Autoclaved soil is represented in light pink (st) and wild one (not autoclaved; “w”) 309 in green. Different letters indicate significant differences among treatments (p < 0.05). 310 We found a significant effect of the identity of the native species competing with S. 311 inaequidens on its performance (ANOVA: Species F = 27.11, p < 0.001; Fig. 2; 312 Supplementary information, Table S4). Trifolium repens was not considered in the 313 analysis because the species only survived in one pot. Senecio inaequidens had the 314 smallest individuals when competing with Wahlenbergia androsacea (SA community) 315 and the largest when competing with Hibiscus trionum and Bromopsis erecta (SA and IT 316 community, respectively). 317 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 16 318 Figure 2. Variation in Senecio inaequidens height based on the identity of the 319 competing species in the community (ANOVA: Species F = 27.11, p < 0.001). IT 320 represents the naïve community from the invasive range (in purple) and SA is the 321 experienced community from the native range (in turquoise). Different letters indicate 322 significant differences among treatments (p < 0.05). 323 Senecio performance in relation to phylogenetic distance from the native community 324 When exploring the effect of phylogenetic distance between the native species and S. 325 inaequidens, we applied two measures of phylogenetic distance. In the first one, we 326 weighted the abundances of native species. We first eliminated an outlier here since 327 distances were above 1.5 times the interquartile range. This number resulted from the 328 dominance of one native species and the mortality of all other natives in one 329 community. After this procedure, we did not find a significant effect of weighted 330 phylogenetic native-invasive distances (CWMPD) on either maximum height or lateral 331 growth (LM: R-squaredheight = -0.08, p = 0.91, R-squaredlat. growth = -0.08, p = 0.88; Fig. 3; 332 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 17 Supplementary information, Table S5). For the second measure of phylogenetic 333 distance, the distance of the most abundant native species in each community to the 334 invasive S. inaequidens (DMANS), we found a significant relationship with the height of 335 S. inaequidens (R-squared = 0.32, p = 0.02; Fig. 3.; Supplementary information, Table 336 S5), though for lateral growth the effect was not significant (R-squared = 0.19, p = 0.06; 337 Supplementary information, Table S4). Wahlenbergia undulata and Wahlenbergia 338 androsacea (SA community) were the most phylogenetically similar species to S. 339 inaequidens (Supplementary information, Fig. S1). 340 341 Figure 3. Relationship between the height of Senecio inaequidens and two measures of 342 phylogenetic distance between S. inaequidens and the native species: (left panel) 343 phylogenetic distance weighted by species abundance (CWPD; R-squared = -0.07, p = 344 0.78); (right panel) phylogenetic distance of the most abundant native species in each 345 community (DMANS); R-squared = 0.32, p = 0.02). IT represents the naïve community 346 from the invasive range (in purple) and SA is the experienced community from the 347 native range (in turquoise). 348 349 350 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 18 Soil bacterial community and its influence on Senecio performance 351 The analysis of soil bacterial communities yielded a total of 687,210 valid sequences, 352 ranging between 2293 and 199,736 per sample, from which 8471 ASVs were inferred. 353 At phylum level, 46.8 ± 6.1% of sequences were classified as Pseudomonadota, 20.7 ± 354 7.1% as Actinomycetota, and 10.6 ± 3.1% as Bacteroidota (Supplementary information, 355 Table S6). At genus level, 60.0 ± 9.6% of sequences could not be classified. 356 Unclassified Bacteria were particularly abundant (9.5 ± 2.7%), as well as unclassified 357 members of classes, Beta- and Gammaproteobacteria (5.4 ± 2.8% and 4.8 ± 2.0%, 358 respectively). The most abundant classified genus was Streptomyces, with 3.9 ± 3.4% 359 of average abundance (Supplementary information, Table S7). The NMDS analysis had 360 a stress coefficient under 0.2 at two dimensions (0.128), thus indicating that this number 361 of dimensions in a plot was a good representation of our data (Clarke 1993). The NMDS 362 plot showed a clear separation of the samples of autoclaved soil in pots where S. 363 inaequidens was growing alone (CTR; Fig. 4. Panel A). Samples from the experienced 364 community (SA) tended to spread more, while samples from the naïve community (IT) in 365 any soil condition clustered more closely (Fig. 4. Panel A). Our PERMANOVA test 366 accounted for 47.27% of the overall variation and indicated an effect of both competing 367 communities and soil, but not their interaction, on ASV community structure 368 (PERMANOVA, Community F = 0.23378, p = 0.001, Soil F = 0.11063, p = 0.003; 369 Supplementary information, Table S8). 370 371 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 19 372 Figure 4. Panel A: Two-dimensional plot of our non-metric multidimensional scaling 373 analysis (NMDS) for bacterial ASVs. The control treatment CTR is shown in light blue, 374 the naïve community from the invasive range IT in purple, and the experienced 375 community in the native range SA in turquoise. Autoclaved soil treatment is represented 376 with filled circles (st) and wild soil (not autoclaved) with filled triangles (w). Panel B: 377 Differences among community and soil treatments in ASV richness and Shannon index 378 of soil bacterial communities. Autoclaved soil is represented in light pink and wild soil 379 (not autoclaved) in green. ASV richness: Interaction, p < 0.05; Shannon index (ANOVA, 380 Community F = 5.699, p < 0.05, Soil F = 24.082, p < 0.001, Interaction F = 4.254, p = 381 0.04). 382 383 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 20 We found that the highest richness and diversity (in terms of Shannon index) of ASVs 384 occurred in wild soils from the South African communities and the lowest in the 385 autoclaved soil with no competing species (control). There was a significant interaction 386 between competing community types and soil conditions for bacterial ASVs (GLM, p < 387 0.05, ANOVA, p < 0.05; Supplementary information, Table S9). Specifically, the effect 388 that community had on ASV richness was modified by soil conditions, with less ASV 389 richness and diversity in controls and South African communities growing in autoclaved 390 soil, compared to wild conditions. Furthermore, ASV richness and diversity in Italian 391 communities did not differ between soil conditions (Fig. 4. Panel B). 392 There was a statistically significant relationship between both ASV richness and 393 Shannon index and the height and lateral growth of S. inaequidens (LM height: Adj-394 R2(ASV richness) = 0.32, p < 0.01, Adj-R2(ASV Shannon) = 0.18, p = 0.04, LM lateral 395 growth: Adj-R2(ASV richness) = 0.39, p < 0.01, Adj-R2 (ASV Shannon) = 0.14, p = 396 0.067; Fig. 5, Supplementary information, Table S10). Specifically, S. inaequidens 397 individuals were taller and wider when growing in pots with lower soil bacterial diversity. 398 399 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 21 400 Figure 5. Linear model results for the relationship between soil bacterial diversity, 401 represented as ASV richness and Shannon index, and height and lateral growth of 402 Senecio inaequidens (LM height: Adj-R2(ASV richness) = 0.32, p < 0.01, Adj-R2(ASV 403 Shannon) = 0.18, p = 0.04, LM lateral growth: Adj-R2(ASV richness) = 0.39, p < 0.01, 404 Adj-R2(ASV Shannon) = 0.14, p = 0.067). For reference, the identity of the competing 405 communities is indicated in different colors. 406 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 22

407 With our experiment, we investigated how the eco-evolutionary experience of species in 408 the resident community and the soil biotic conditions influence the performance of 409 Senecio inaequidens. As hypothesized, the identity of competing plant communities 410 significantly affected S. inaequidens growth, supporting the hypothesis that competition 411 with naïve species in the invasive range is less intense than with experienced species 412 from the native range. We only found partial support for our other hypotheses. 413 Specifically, the effect of phylogenetic relatedness in explaining S. inaequidens 414 performance was mixed, with no effect of community-wide distances but a significant 415 influence of the most abundant species relatedness to S. inaequidens. Although the 416 competitive responses of S. inaequidens to the plant communities were not significantly 417 affected by autoclaving the soil in which they grew, soil bacterial diversity still seems to 418 play a role in its performance. 419 Eco-evolutionary experience and species identity modulates competition 420 Our results align with previous studies suggesting that naïve species in the invasive 421 range may lack evolved resistance or competitive strategies against introduced species 422 with which they have had no similar interactions in their evolutionary history (Callaway et 423 al. 2011; Saul et al. 2013; Zhang et al. 2018). For instance, in a removal experiment, 424 Callaway et al. (2011) found Centaurea stoebe L. populations in their native range 425 (Europe) to exhibit a significantly higher response (6.5- to 7.5-fold) to the removal of 426 neighboring plants compared to populations in their invasive range (North America). The 427 reduced competitive effects associated with the lack of eco-evolutionary experience of 428 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 23 the Italian communities could be attributed to several mechanisms, including differences 429 in resource acquisition with naïve native competitors. On the other hand, S. inaequidens 430 may be exerting a stronger competitive response, possibly through allelopathic effects 431 that naïve species have not yet adapted to counter. Additionally, Senecio inaequidens is 432 known to contain secondary metabolites in its tissues (i.e. pyrrolizidine alkaloids) that 433 are poisonous to some animals (Dimande et al. 2007). Invasive populations may benefit 434 from this chemical defense, as naïve herbivores in the newly colonized environment are 435 unlikely to feed on it, further enhancing its invasion success (Scherber et al. 2003; 436 Misuri et al. 2020). Alternatively, its success could be linked to a subtle temporal 437 advantage, allowing it to grow slightly faster and establish dominance earlier in the 438 competition. Indeed, Delory et al. (2019) found S. inaequidens to exhibit strong 439 competitive effects on native plants when it has a temporal advantage due to, for 440 example, the slower growth of competing native species (Delory et al. 2019). 441 We also found that the identity of the species in the community affected S. inaequidens 442 performance. In particular, the South African Wahlenbergia androsacea had a 443 consistent negative effect on S. inaequidens height when present in the community. 444 This pattern was not consistent across species from the native range, indicating that 445 origin or co-occurrence per se is not a strong indicator of competitive effects of the 446 native species. Instead, the traits of the competing species may play a more significant 447 role. For example, a study modeling experimentally derived competitive impact and 448 responses of Acroptilon repens, a species native to Uzbekistan and invasive in North 449 America, found them to be rather dependent on the traits of the species it was 450 competing with (Xiao et al. 2013). These results together support the idea that invasion 451 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 24 success and impact are shaped by both the introduced species traits and the 452 adaptability and competitiveness of the recipient community. 453 Our phylogenetic analyses support the idea that the presence of a dominant, closely 454 related native species (e.g., Wahlenbergia sp.) may increase the competitive resistance 455 against S. inaequidens, supporting Darwin’s naturalization hypothesis. This finding is in 456 line with the assumption that phylogenetic relatedness can be a good proxy for 457 functional trait similarity and resource use overlap, leading to more intense competition 458 (Divíšek et al. 2018). Our findings also align with previous research showing that biotic 459 resistance in native plant communities against other invasive Asteraceae species in 460 Europe, such as Ambrosia artemisiifolia L. and Solidago gigantea Aiton, is strongly 461 influenced by phylogenetic proximity to dominant native species (Yannelli et al. 2017). 462 Therefore, while community phylogenetic similarity may not strongly predict invasion 463 success (Dostál 2011), interactions with key species within the community, particularly 464 the most abundant ones, may play a critical role. Interestingly, a recent observational 465 study carried out in Northern Italy described a negative relationship between S. 466 inaequidens performance and phylogenetic similarity to resident species in the field 467 (Quaglini et al. 2025), lending support to what is known as the pre-adaptation 468 hypothesis. The study found that S. inaequidens performed better when growing 469 alongside more similar species, particularly in more productive habitats. Such 470 apparently contradictory results could be reconciled by recent reviews suggesting that 471 Darwin’s naturalization and pre-adaptation hypotheses are not mutually exclusive, but 472 may operate at different spatial scales (Thuillier et al. 2010; Ma et al. 2016). Namely, 473 successful alien species would be more closely related to natives at broader spatial 474 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 25 scales, due to environmental filtering, but more distantly related at finer spatial scales, 475 where competition for limiting resources becomes more important (Ma et al. 2016). In 476 other words, at large scales, environmental filtering selects for invaders that are adapted 477 to the conditions of the new area, while at small spatial scales, the role of competition 478 for limiting resources becomes more important. This highlights the context dependency 479 of biotic resistance, where competition dynamics can shift depending on environmental 480 conditions and resource levels. 481 Reduced soil bacterial diversity benefits Senecio performance under competition 482 We observed distinct proportions of the most abundant bacterial phyla across 483 treatments, mainly Pseudomonadota and Actinomycetota, with South African soils 484 exhibiting slightly higher levels of Actinomycetota, while Italian soils had more 485 Pseudomonadota. Actinomycetota, a highly diverse and globally widespread bacterial 486 phylum (van Bergeijk et al. 2020), along with Pseudomonadota, is commonly found 487 across various habitats in Europe (Labouyrie et al. 2023). Autoclaved soil showed a 488 significantly lowered bacterial diversity compared to wild soil, at least in the control and 489 South African communities. In those conditions, bacterial communities could not recover 490 their original diversity after the sterilizing treatment which eliminated to some degree the 491 existing soil microbial community. The composition of the plant community competing 492 with S. inaequidens also affected soil microbial diversity, with the South African 493 communities supporting higher bacterial diversity in wild soil compared to other 494 treatments. Control pots with S. inaequidens individuals growing alone maintained the 495 most unique bacterial communities, especially in autoclaved soils, whereas soils with 496 competing native plants showed greater similarity in community structure. For instance, 497 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 26 control pots with autoclaved soil were characterized by a generally higher abundance of 498 Nocardioides (10.0% on average) compared to the other treatments. Such results 499 suggest that plant community identity influences microbial assemblages, even after a 500 sterilization treatment. One possible explanation for the observed patterns is that 501 introduced plants like S. inaequidens may bring along their associated bacteria (e.g. in 502 the seeds), which can aid their invasion by enhancing establishment, nutrient 503 acquisition, growth, or resistance to local biotic pressures (van der Putten et al. 2007; Le 504 Roux et al. 2017; Zhang et al. 2023). 505 Soil autoclaving did not have a significant direct effect on the overall competitive 506 response of S. inaequidens to competition, rather bacterial diversity was found to 507 influence its performance. Autoclaved soils generally supported reduced bacterial 508 diversity, and lower bacterial diversity was associated with increased S. inaequidens 509 height. This finding is somewhat unexpected, given that higher microbial diversity is 510 typically associated with ecosystem stability and resilience (Ehrenfeld 2003; Wardle et 511 al. 2004). One possible explanation, consistent with our soil autoclaving results, is that 512 reduced microbial diversity may lower the presence or activity of pathogens and 513 competitors, thereby enabling S. inaequidens to allocate more resources toward growth. 514 This aligns with the enemy release hypothesis, which posits that invasive species may 515 escape their natural enemies in new environments, reducing their biotic resistance and 516 enhancing their performance (Keane and Crawley 2002; Heger et al. 2024). The 517 enormous diversity of soil microbial communities can harbor generalist pathogens that 518 affect invasive plants but also disadvantage native species through pathogen spillover, 519 especially if exotics are more tolerant (van der Putten et al. 2007; Dawson and Schrama 520 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 27 2016). Therefore, another possible explanation for the increased height of S. 521 inaequidens in soils with low bacterial diversity is that native plants may be less vigorous 522 or competitive under these conditions, possibly due to a shortage of beneficial microbes 523 or disruptions of commensalistic and symbiotic relationships between the soil microbial 524 community and the plants. With less competition from native plants, S. inaequidens 525 could allocate more resources to growth, leading to taller individuals. This is supported 526 by other research showing correlations between S. inaequidens traits, particularly those 527 related to competitive ability and resource allocation, and bacterial diversity (e.g. 528 Thébault et al. 2010). These findings suggest that shifts in soil microbial diversity could 529 influence S. inaequidens ability to outcompete native species, potentially by altering 530 nutrient availability, pathogen pressure, or the presence of beneficial microbial partners. 531 It is important to note that methods like autoclaving can alter soil chemistry, nutrient 532 availability, and physical structure, potentially confounding experimental results by 533 affecting both microbial communities and abiotic factors (Perkins et al. 2013). We note 534 that sterilization does not fully eliminate bacterial DNA, however, its influence is likely 535 minimal, as samples were collected when community shifts dominate and residual DNA 536 from cells killed ~90 days earlier is probably negligible. Finally, the 84-day duration of 537 the experiment provided valuable insights, though longer-term studies could offer a 538 more comprehensive understanding of plant-soil feedbacks and competitive dynamics 539 (Liu et al. 2024). 540 541 542 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 28

and implications for management 543 Our study highlights the interplay between eco-evolutionary experience, plant 544 phylogenetic relationships and soil biotic conditions. By analysing the interactions 545 between this invasive species and plant communities from both its native and invasive 546 ranges, we provide insights into the possible mechanisms driving its invasion success, 547 which seems to be favoured by the inexperience of the community of the invasive range 548 with respect to the invader (i.e. naivety). Based on our findings, we argue that selecting 549 few phylogenetically related species at high abundances to outcompete S. inaequidens 550 could be a promising practice for management in areas under restoration. In particular, 551 the observation that S. inaequidens performs better in the presence of naïve species 552 and lower microbial diversity indicates that restoration efforts might benefit from 553 enhancing the competitive ability of native species and promoting microbial diversity. 554 This could involve the selection of native species that are closely related to the invader 555 or have strong competitive abilities and testing soil amendments to increase microbial 556 diversity and resilience. Furthermore, our findings suggest that management strategies 557 should also consider the composition and functional roles of native communities by 558 selecting multi-species suites of closely related competitors displaying similar trait 559 profiles, as well as the structure of soil microbial communities. 560

561 FAY acknowledges funding from the Feodor Lynen Fellowship, awarded by the 562 Alexander von Humboldt Foundation, and the Rising Star Fellowship, granted by the 563 Department of Biology, Chemistry, and Pharmacy at Freie Universität Berlin. 564 Author-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941 29

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