{"paper_id":"0503cd8a-044d-45e8-ad04-cd6ca5ed28b1","body_text":"PREPRINT\nAuthor-formatted, not peer-reviewed document posted on 03/06/2025\nDOI: https://doi.org/10.3897/arphapreprints.e160941\nDon’t be naïve: Eco-evolutionary experience better\nexplains invasion success of Senecio inaequidens than\nsoil conditions\nLara A. Quaglini,  Florencia A Yannelli, Isabella Gandolfi, Andrea Franzetti, Sarah Caronni,  Chiara\nMontagnani,  Clinton Carbutt,  Jonathan M. Jeschke,  Sandra Citterio,  Rodolfo Gentili\n\n1 \n \nDon’t be naïve: Eco-evolutionary experience better explains invasion success of 1 \nSenecio inaequidens than soil conditions 2 \nLara A. Quaglini1,2*, Florencia A. Yannelli3,4,5*, Isabella Gandolfi2, Andrea Franzetti2, 3 \nSarah Caronni2, Chiara Montagnani2, Clinton Carbutt6,7, Jonathan M. Jeschke3,4, 4 \nSandra Citterio2, Rodolfo Gentili2 5 \n* These authors contributed equally to this work 6 \n1 Natural History Museum of Milan, Corso Venezia 55, 20121, Milan, Italy 7 \n2 Department of Earth and Environmental Sciences, Università degli Studi di Milano-8 \nBicocca, Piazza della Scienza 1, 20126 Milan, Italy  9 \n3 Department of Biology, Chemistry, Pharmacy, Institute of Biology, Freie Universität 10 \nBerlin, Königin-Luise-Str. 1-3, 14195 Berlin, Germany 11 \n4 Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), Müggelseedamm 12 \n310, 12587 Berlin, Germany 13 \n5 Argentine Institute for Dryland Research, CONICET and Universidad Nacional de 14 \nCuyo, Av. Ruiz Leal s/n, 5500 Mendoza, Argentina 15 \n6 School of Life Sciences, University of KwaZulu-Natal, Scottsville 3209, South Africa  16 \n7 Scientific Services, Ezemvelo KZN Wildlife, Cascades 3202, South Africa 17 \nCorresponding author: Florencia A. Yannelli, florenciayannelli@gmail.com 18 \n 19 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n2 \n \nAbstract 20 \nIntroduced species encounter novel biotic and abiotic conditions that influence their 21 \nsuccess in new environments. Their advantage is often linked to reduced competition 22 \nfrom native species that lack eco-evolutionary experience, as well as to their ability to 23 \npre-empt resources. Once established, their success can also be shaped by changes in 24 \nsoil conditions, particularly through interactions with soil microbial communities. 25 \nUnderstanding how these factors influence invasion success can provide valuable 26 \ninsights into predicting future invasions under global change. In this study, we examined 27 \nhow eco-evolutionary experience and soil bacterial communities influenced the 28 \nperformance of the invasive subshrub Senecio inaequidens DC. We conducted a fully 29 \nfactorial experiment in growth chambers consisting of two factors: competing community 30 \nidentity with three levels (plant species from its native range (South Africa), from its 31 \ninvasive range (Italy) and a control with only S. inaequidens) and soil biota conditions 32 \nwith two levels (wild soil and autoclaved soil with lower microbial load). Our results 33 \nshowed that plant community identity had the strongest effect on S. inaequidens growth 34 \n(height and lateral spread), with the smallest individuals occurring in competition with 35 \nSouth African species. Growing on autoclaved soil had no major impact on plant height, 36 \nsuggesting that reduced competition played a greater role than soil bacterial differences 37 \nin determining plant performance. Suppression was stronger when the competing native 38 \nspecies were more closely related to S. inaequidens. Soil bacterial communities were 39 \ninfluenced by both plant identity and soil treatment, and S. inaequidens performed 40 \nbetter in soils with lower bacterial diversity, possibly due to reduced pathogen pressure. 41 \nThese findings suggest that invasive species management could be improved by 42 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n3 \n \nfostering competition with evolutionarily experienced native species and maintaining or 43 \nenhancing soil microbial diversity to limit invader success. 44 \nKeywords: eco-evolutionary experience; phylogenetic similarity, plant traits; relatedness; 45 \nsoil bacteria; South African ragwort 46 \n 47 \n 48 \n 49 \n 50 \n 51 \n 52 \n 53 \n 54 \n 55 \n 56 \n 57 \n 58 \n 59 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n4 \n \nIntroduction 60 \nUpon entering new environments, introduced species encounter novel biotic and abiotic 61 \nconditions that can either facilitate or hinder the invasion process (Heger et al. 2019). 62 \nTo succeed, these species must effectively establish new interactions, make use of and 63 \ncompete for available resources to eventually become invasive (Funk and Vitousek 64 \n2007). For example, introduced plants may encounter new competitive, pathogenic or 65 \nherbivore pressures (Funk et al. 2008), lose critical mutualistic interactions (Mitchell et 66 \nal. 2006) or face novel traits such as allelochemical release, to which they lack eco-67 \nevolutionary experience (Callaway and Ridenour 2004). Understanding the mechanisms 68 \nunderlying the success of species introduced in new areas can provide valuable insights 69 \ninto predicting other processes, such as the expansion of species ranges and potential 70 \nfuture invasions in the context of global change (Fristoe et al. 2021). 71 \nThe outcome of novel interactions between introduced and native species may be 72 \nshaped by their evolutionary history, particularly their past experiences interacting with 73 \nspecific species or traits (Saul et al. 2013; Saul and Jeschke 2015). In this context, the 74 \ninvasion success of introduced plants might be hindered if the recipient community 75 \nincludes closely related species, as these species or other community members are 76 \nmore likely to have eco-evolutionary experience with similar competitors, predators, or 77 \nother antagonistic interactions (Saul et al. 2013). Conversely, functionally dissimilar 78 \nintroduced species with novel physiological and morphological traits, such as the ability 79 \nof exudating allelopathic compounds or the ability to exploit untapped resources, may 80 \ngain a competitive advantage in communities where native species lack eco-81 \nevolutionary experience with these traits. This experience can influence how native 82 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n5 \n \nspecies respond to novel competitors, thereby shaping invasion dynamics (Heger and 83 \nTrepl 2003; Heger et al. 2019; Novoa et al. 2020). 84 \nAt the community level, niche overlap and resource preemption due to competition for 85 \nlimited resources, are key factors in the success of introduced plant species 86 \n(MacDougall et al. 2009). Darwin's naturalization hypothesis posits that introduced 87 \nspecies would struggle to establish in communities with closely related native species 88 \nbut have a higher invasion success when they are more phylogenetically distant from 89 \nthe resident flora (Darwin 1859; Daehler 2001; Yannelli et al. 2025). The underlying 90 \nassumption is that niche similarity among species is phylogenetically conserved 91 \n(Prinzing 2001), with phylogenetic relatedness reflecting shared traits that influence 92 \nspecies ability to coexist (Blomberg and Garland 2002). This success is further shaped 93 \nby the species competitive ability; both in terms of its competitive effect, or its capacity 94 \nto suppress other individuals by depleting resources, and its competitive response, or its 95 \nability to tolerate growth suppression from neighboring plants (Goldberg 1990). While 96 \nthe hypothesis has shed light on initial establishment (Park and Potter 2013; Yannelli et 97 \nal. 2017), inconsistencies arise from temporal variations and shifts in traits among 98 \nclosely related species (Burns and Winn 2006; Thuiller et al. 2010; Li et al. 2015). To 99 \nbetter understand the drivers of invasion success, it is crucial to test this hypothesis with 100 \nspecies from both native and invasive ranges, a perspective that has yet to be fully 101 \nexplored (but see e.g. Zheng et al. 2018).  102 \nResource availability and competition can be influenced by belowground dynamics 103 \nlinked to plant traits and microbial communities. Soil organisms play a crucial role in 104 \nmediating interactions between native and invasive plant species, affecting e.g., 105 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n6 \n \ncompetitive, mutualistic, and pathogenic interactions, and ultimately invasion success 106 \n(Abbott et al. 2015; Fahey and Flory 2022). The competitive strength of invasive plants 107 \nmay be increased by altered microbial communities if they lose coevolved specialist 108 \npathogens from their native range (Mitchell and Power 2003; Fahey and Flory 2022) or 109 \nserve as reservoirs for pathogens that disproportionately affect native species (Eppinga 110 \net al. 2006; Mangla and Callaway 2008). New interactions with pathogenic or mutualistic 111 \nmicroorganisms can be established with invasive species in the new area based on 112 \nsimilarities in traits to those of native species, even without any previous evolutionary 113 \nexperience (Eppinga et al. 2006; Diez et al. 2010). Trait differences in invasive plants 114 \ncompared to the recipient communities, particularly those associated with acquisitive 115 \nstrategies, can also lead to shifts in the microbial community from fungi- to bacteria-116 \ndominated (Ehrenfeld 2003; Wardle et al. 2004; Torres et al. 2021). Nevertheless, the 117 \nrole of soil bacteria in mediating competition and plant community assembly, in 118 \nparticular through pathogenic interactions, seems highly limited (van der Putten et al. 119 \n2007; Dawson and Schrama 2016). 120 \nWe selected the invasive plant Senecio inaequidens DC., commonly known as “South 121 \nAfrican ragwort” or “Canary Weed”, as our study species (hereafter sometimes referred 122 \nto just with its generic name). This perennial chamaephyte native to South Africa, and 123 \ninvasive in areas of the country outside of its native range, was introduced to Europe in 124 \nthe late 19th century (Ernst 1998) and has become invasive in disturbed areas. Senecio 125 \ninaequidens has been found to produce allelopathic defenses in the form of pyrrolizidine 126 \nalkaloids (Joosten and van Veen 2011), which can protect it against both above- and 127 \nbelowground herbivory (Caño et al. 2009; Thoden et al. 2009), influencing soil microbial 128 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n7 \n \ncommunities (Harkes et al. 2017). Senecio inaequidens traits were also found to 129 \ncorrelate with rhizosphere biota, with bacterial diversity being positively associated with 130 \nresource allocation to belowground growth and late flowering (Thébault et al. 2010). 131 \nFurthermore, Van De Walle et al. (2022) found S. inaequidens to modify soil abiotic 132 \nconditions, increasing nutrient concentrations via litter deposition and eliciting increased 133 \ngrowth of co-occurring native species in nutrient-poor habitats. However, the impact of 134 \nthese traits and soil community alterations on its competitive success in the invasive 135 \nversus native range has remained unexplored. 136 \nIn this study, we investigated S. inaequidens competitive response to native plant 137 \nspecies from both its native and invasive ranges under controlled experimental 138 \nconditions. The experimental communities included species with which S. inaequidens 139 \nshares a history of eco-evolutionary interactions (native range) and species to which it is 140 \nevolutionarily naïve (invasive range), lacking such historical associations. We also 141 \nexamined how soil conditions, including the presence or absence (by autoclaving) of 142 \nsoil biota from the invasive range, influenced these competitive interactions. 143 \nSpecifically, we explored whether eco-evolutionary experience, phylogenetic 144 \nrelatedness, and soil biota could explain Senecio's performance when competing with 145 \nnative plant communities. We hypothesized that: (i) S. inaequidens performance will 146 \ndepend on the identity of the competing plant communities, and it will perform better 147 \nwhen competing with naïve species from the invasive range compared to experienced 148 \nspecies from the native range; (ii) an increase in phylogenetic relatedness between S. 149 \ninaequidens and the competing species in the community will result in lower 150 \nperformance of S. inaequidens, following Darwin’s naturalization hypothesis; and (iii) 151 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n8 \n \nsoil biota will influence the competitive interactions between S. inaequidens and native 152 \nplant communities, with reduced biotic effects in autoclaved soils leading to better 153 \nperformance of S. inaequidens compared to non-autoclaved soils. 154 \nMaterials and Methods 155 \nPlant and soil material collection 156 \nSenecio inaequidens is a subshrub that belongs to the Asteraceae family, often 157 \nreaching 40-100 cm in height. Native to South Africa's highlands, it was introduced to 158 \nEurope as a wool contaminant (Ernst 1998). It thrives in disturbed areas like roadsides, 159 \nrailways, and quarries, as well as dry grasslands, pastures, and vineyards (Heger and 160 \nBöhmer 2005; López-García and Maillet 2005). In its native range, the species exists in 161 \ndiploid (2n = 20) and tetraploid (2n = 40) forms, but only tetraploids are found in Europe 162 \n(Lafuma et al. 2003).  163 \nSeeds of S. inaequidens were collected from a population located in the former quarry 164 \nof Collepedrino, Northern Italy, which is currently heavily invaded by this species 165 \n(Bergamo, 45°46'37.4\"N 9°31'09.5\"). To design the competing native communities, we 166 \nchose five species known to co-occur with S. inaequidens in each range (i.e., native and 167 \ninvasive). To assess this in the native range of the invasive species (i.e., South Africa), 168 \nwe used the National Collections database (http://posa.sanbi.org/) to select species 169 \ndocumented in the area where tetraploid populations of S. inaequidens have been 170 \nreported (Lafuma et al. 2003). Upon availability in local seed companies, we refined the 171 \nlist of native species by a second check against the results of vegetation surveys (Du 172 \nPreez and Bredenkamp 1991), to be sure that all natives would have co-occurred in a 173 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n9 \n \nplot-sized area. As a result, we selected a multi-species suite comprising Aristida 174 \ncongesta Roem. & Schult. (Poaceae), Hibiscus trionum L. (Malvaceae), Salvia disermas 175 \nL. (Lamiaceae), Wahlenbergia androsacea A. DC. and Wahlenbergia undulata (L.f.) A. 176 \nDC. (Campunulaceae) for the native range. Seed material for the native range was 177 \npurchased at the local seed company Silverhill (Cape Town, South Africa). In the 178 \ninvasive range (i.e. Italy), we selected native species according to known co-occurrence 179 \nin the Collepedrino quarry (Gentili et al. 2020) and collected seeds in the same area. 180 \nThe list included Bromopsis erecta (Huds.) Fourr. (Poaceae), Hypericum perforatum L. 181 \ns.l. (Hypericaceae), Onobrychis viciifolia Scop. (Fabaceae), Poterium sanguisorba L. s.l. 182 \n(Rosaceae) and Trifolium repens L. (Fabaceae). We performed germination tests for all 183 \nnative species to find the best conditions for their germination (Supplementary 184 \nInformation, Section S1, Tab. S1).  185 \nSoil used for the experiment was collected in the same quarry. It was placed in open dry 186 \nbags and stored at room temperature until setting the experiment. We prepared the 187 \nexperimental substrate by mixing the quarry soil, which was highly rocky, with common 188 \npotting substrate (TERCOM potting soil) in a 1:1 ratio to favor plant growth under 189 \ncontrolled conditions (growth chamber). Before setting the pot experiment, we 190 \nautoclaved half of this soil mix at 120°C for 45 minutes. 191 \nExperimental design and setting 192 \nOur experiment consisted of a fully factorial design with a combination of two factors: 193 \ncompeting community and soil biota. The competitive communities’ identity had three 194 \nlevels, namely species from the native range considered to be experienced (SA; South 195 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n10 \n \nAfrica), species from the invasive range considered to be naïve (IT; Italy) and the 196 \ncontrol (CTR) with S. inaequidens individuals growing alone. Soil biota conditions had 197 \ntwo levels, i.e. autoclaved (st) and non-autoclaved soil (w; henceforth “wild”). Each 198 \ntreatment combination was replicated five times, making up a total of 30 experimental 199 \npots. Before the experiment, all seeds were stratified by placing them in paper bags at 200 \n4°C for about 1 month. Two different methods of germination were used to ensure the 201 \nsurvival of the seeds and high germination rates, according to results of germination 202 \ntests performed before the experiment: (1) Directly in plastic cups with a mix of 203 \nautoclaved common potting soil and sand with a 1:1 ratio; (2) in Petri dishes with 204 \nmoistened filter paper, which were transplanted to plastic cups filled with autoclaved 205 \ncommon potting soil and sand at a 1:1 ratio, a few days after germination (see protocols 206 \nin Supplementary information, Section S1). When the seedlings were about 20 days old, 207 \nwe placed two individuals of the invasive S. inaequidens in the middle of 2L pots filled 208 \nwith a mix of quarry soil and common potting soil. At the same time, in all treatment 209 \ncombinations that required competition with natives, we added 5 individuals for each 210 \nnative species distributed at the edges of the pot. We then completely randomized the 211 \npots and placed them in a growth chamber with an average temperature of 29°C, 212 \nrelative humidity of 42%, and a day-night cycle of 14 and 10 hours, respectively. These 213 \nvalues were consistent with the growth conditions of S. inaequidens when invading 214 \nruderal dry habitats (railways, roadsides, etc.). Plants were watered every other day for 215 \nthe first days, and twice a week for the rest of the experiment. The experiment ran for 216 \n84 days, when some S. inaequidens individuals started to die. 217 \n 218 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n11 \n \nMeasurements and data processing 219 \nAfter 84 days from the start of the experiment, we collected data on plant vegetative 220 \nfitness and survivorship, as a proxy for success. Specifically, we measured the 221 \nmaximum height as the shortest distance between the upper boundary of the main 222 \nphotosynthetic tissues on a plant and the ground level, and the lateral growth of each 223 \nindividual of S. inaequidens as the maximum width of the canopy (Pérez-Harguindeguy 224 \net al. 2013). At this point, we also recorded the number and identity of the native 225 \nspecies that survived in each pot.  226 \nTo assess the effect of relatedness on S. inaequidens performance, we calculated the 227 \nphylogenetic distances among all species in our experiment from a phylogenetic tree for 228 \nangiosperms as a backbone (Zanne et al. 2014) that was pruned from all species that 229 \nwere not included in our experiment (Supplementary information, Fig. S1). We then 230 \ncalculated community-weighted phylogenetic distances to the invader (CWMPD) by 231 \nweighting the native community-invasive distances with the proportion (based on the 232 \nnumber of individuals alive) of each species (in terms of number of individuals) in the 233 \npot. Further, we also obtained the distance of the most abundant species in each 234 \ncommunity to the invader (DMANS) to examine the effect of these species on S. 235 \ninaequidens growth. In the case of more than one species dominating the community in 236 \nthe same abundance, we used total mean phylogenetic distances to every dominant 237 \nnative. To characterize the soil bacterial communities in each treatment combination, 238 \nwe collected soil samples at the end of the experiment (after 84 days) from three 239 \nrandomly selected pots (n = 18). The samples were stored at -20°C until processing. 240 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n12 \n \nDNA extraction and Next Generation sequencing 241 \nGenomic DNA was extracted using the FastDNA® Spin Kit for Soil (MP Biomedicals, 242 \nSolon, OH, USA) following the manufacturer’s instructions. A first PCR amplification 243 \nwas carried out using the 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 519R (5’-244 \nGWATTACCGCGGCKGCTG-3’) primers (Frank et al. 2008; Hollister et al. 2011) on the 245 \noriginal DNA extract and on the 1:10, 1:100, 1:1000 and 1:10000 dilutions, to detect the 246 \npossible presence of PCR inhibitors. Amplification conditions were: initial denaturation 247 \nat 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 \nfinal extension at 72°C for 5 min. A second PCR was then performed using 783F and 249 \n1046R primers on the V5-V6 hypervariable regions of the bacterial 16S rRNA gene, with 250 \ncustomized oligonucleotide barcodes (6bp, see sequence in Table S2) added to their 5’ 251 \nend (Gandolfi et al. 2024). We used GoTaq® Green Master Mix (Promega Corporation, 252 \nMadison, WI, USA) and 1 µM of each primer, for a final volume of 2 x 50 µL for each 253 \nsample. This second amplification was performed under the following conditions: initial 254 \ndenaturation at 94°C for 4 min, 28 cycles at 94°C for 50 s, 47°C for 30 s and 72°C for 255 \n30 s, and a final extension at 72°C for 5 min. The PCR products were purified using the 256 \nWizard® SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, 257 \nUSA), following the manufacturer's instructions, and the DNA content was quantified 258 \nwith the Qubit ® 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). Amplicon 259 \nlibraries were prepared with nine samples each, identifiable due to different barcode 260 \npairs. Library preparation with the addition of standard Nextera indices (Illumina, Inc., 261 \nSan Diego, CA, USA) and sequencing with the MiSeq Illumina platform (Illumina, Inc., 262 \nSan Diego, CA, USA), using a 2 × 250 bp paired-end protocol, was performed at the 263 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n13 \n \nConsorzio per il Centro di Biomedicina Molecolare (CBM), located in Trieste, Italy. 264 \nAmplicon Sequence Variants (ASVs) were inferred through the DADA2 algorithm 265 \n(Callahan et al. 2016), as described in Gandolfi et al. (2024). 266 \nData analysis 267 \nAll statistical analyses were performed using R version 4.3.1 (R-Core-Team 2023) and 268 \nthe vegan package (Oksanen et al. 2022), unless stated otherwise. We used two-way 269 \nANOVA to assess if the average height and lateral growth of S. inaequidens were 270 \naffected by the competing community, soil conditions and their interaction. Since there 271 \nwas an imbalance in the experimental replication due to the mortality of S. inaequidens 272 \nin some replicates, we used the Type III test. We then performed post-hoc pairwise 273 \ncomparisons with Tukey tests. In the same way, we used one-way ANOVA to test the 274 \neffect of native species identity on the height of S. inaequidens to explore the impact of 275 \nthe presence of individual species. To evaluate the effect of CWMPD and DMANS in 276 \neach community on average S. inaequidens height and lateral growth, we used linear 277 \nregressions.  278 \nWe used Non-metric Multidimensional Scaling (NMDS) analysis based on Bray-Curtis 279 \ndissimilarity distances (Bray and Curtis 1957) to visualize differences in soil bacterial 280 \ncommunity structure according to the treatments using the metaMDS function. We 281 \ncarried out a PERMANOVA test using the adonis2 function to assess treatment 282 \ncombination effects on soil bacterial communities. Before performing these multivariate 283 \nanalyses, we transformed the bacterial ASV abundance matrix with Hellinger distance 284 \nto reduce the emphasis on ASV abundances while highlighting their presence or 285 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n14 \n \nabsence and mitigate the double-zero issue when comparing ASV compositions across 286 \nsamples (Bocard et al. 2018). We calculated ASV richness and Shannon index for each 287 \ntreatment combination on the rarefied bacterial data, which were based on the sample 288 \nwith the lowest reading depth (2293). We then evaluated the effects of our treatments 289 \non ASV richness using generalized linear models with a quasi-Poisson distribution to 290 \ncorrect for overdispersion present in the data (Cameron and Trivedi 1990) and used 291 \nANOVA for the Shannon index. Finally, we explored the effect of bacterial alpha-292 \ndiversity, i.e., ASV richness and Shannon index, on the height and lateral growth of S. 293 \ninaequidens using a linear model. 294 \nResults 295 \nEffect of competition and soil biota conditions on Senecio performance  296 \nSenecio inaequidens performance was affected by the community it was growing along 297 \nwith more than soil conditions, compared to the control treatment in pots where it grew 298 \nwithout competition. Specifically, in terms of S. inaequidens maximum height at day 84, 299 \nonly community identity had a significant effect (ANOVA: Community: F = 4.31, p < 300 \n0.03; Fig.1; Supplementary information, Table S3). We did not find an effect of 301 \ncommunity and soil conditions on lateral growth (ANOVA p > 0.05; Fig. 1). 302 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n15 \n \n 303 \nFigure 1. Differences among community and soil treatments in height (left panel) and 304 \nlateral growth (right panel) of Senecio inaequidens 84 days after the experiment started. 305 \nCTR represents the control treatment with no native species growing with Senecio, IT is 306 \nthe naïve community from the invasive range in Italy, and SA is the experienced 307 \ncommunity from the native range in South Africa (ANOVA: Community F = 4.31, p < 308 \n0.03). Autoclaved soil is represented in light pink (st) and wild one (not autoclaved; “w”) 309 \nin green. Different letters indicate significant differences among treatments (p < 0.05).    310 \nWe found a significant effect of the identity of the native species competing with S. 311 \ninaequidens on its performance (ANOVA: Species F = 27.11, p < 0.001; Fig. 2; 312 \nSupplementary information, Table S4). Trifolium repens was not considered in the 313 \nanalysis because the species only survived in one pot. Senecio inaequidens had the 314 \nsmallest individuals when competing with Wahlenbergia androsacea (SA community) 315 \nand the largest when competing with Hibiscus trionum and Bromopsis erecta (SA and IT 316 \ncommunity, respectively).   317 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n16 \n \n 318 \nFigure 2. Variation in Senecio inaequidens height based on the identity of the 319 \ncompeting species in the community (ANOVA: Species F = 27.11, p < 0.001). IT 320 \nrepresents the naïve community from the invasive range (in purple) and SA is the 321 \nexperienced community from the native range (in turquoise). Different letters indicate 322 \nsignificant differences among treatments (p < 0.05).    323 \nSenecio performance in relation to phylogenetic distance from the native community  324 \nWhen exploring the effect of phylogenetic distance between the native species and S. 325 \ninaequidens, we applied two measures of phylogenetic distance. In the first one, we 326 \nweighted the abundances of native species. We first eliminated an outlier here since 327 \ndistances were above 1.5 times the interquartile range. This number resulted from the 328 \ndominance of one native species and the mortality of all other natives in one 329 \ncommunity. After this procedure, we did not find a significant effect of weighted 330 \nphylogenetic native-invasive distances (CWMPD) on either maximum height or lateral 331 \ngrowth (LM: R-squaredheight = -0.08, p = 0.91, R-squaredlat. growth = -0.08, p = 0.88; Fig. 3; 332 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n17 \n \nSupplementary information, Table S5). For the second measure of phylogenetic 333 \ndistance, the distance of the most abundant native species in each community to the 334 \ninvasive S. inaequidens (DMANS), we found a significant relationship with the height of 335 \nS. inaequidens (R-squared = 0.32, p = 0.02; Fig. 3.; Supplementary information, Table 336 \nS5), though for lateral growth the effect was not significant (R-squared = 0.19, p = 0.06; 337 \nSupplementary information, Table S4). Wahlenbergia undulata and Wahlenbergia 338 \nandrosacea (SA community) were the most phylogenetically similar species to S. 339 \ninaequidens (Supplementary information, Fig. S1).   340 \n  341 \nFigure 3. Relationship between the height of Senecio inaequidens and two measures of 342 \nphylogenetic distance between S. inaequidens and the native species: (left panel) 343 \nphylogenetic distance weighted by species abundance (CWPD; R-squared = -0.07, p = 344 \n0.78); (right panel) phylogenetic distance of the most abundant native species in each 345 \ncommunity (DMANS); R-squared = 0.32, p = 0.02). IT represents the naïve community 346 \nfrom the invasive range (in purple) and SA is the experienced community from the 347 \nnative range (in turquoise). 348 \n 349 \n 350 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n18 \n \nSoil bacterial community and its influence on Senecio performance 351 \nThe analysis of soil bacterial communities yielded a total of 687,210 valid sequences, 352 \nranging between 2293 and 199,736 per sample, from which 8471 ASVs were inferred. 353 \nAt phylum level, 46.8 ± 6.1% of sequences were classified as Pseudomonadota, 20.7 ± 354 \n7.1% as Actinomycetota, and 10.6 ± 3.1% as Bacteroidota (Supplementary information, 355 \nTable S6). At genus level, 60.0 ± 9.6% of sequences could not be classified. 356 \nUnclassified Bacteria were particularly abundant (9.5 ± 2.7%), as well as unclassified 357 \nmembers of classes, Beta- and Gammaproteobacteria (5.4 ± 2.8% and 4.8 ± 2.0%, 358 \nrespectively). The most abundant classified genus was Streptomyces, with 3.9 ± 3.4% 359 \nof average abundance (Supplementary information, Table S7). The NMDS analysis had 360 \na stress coefficient under 0.2 at two dimensions (0.128), thus indicating that this number 361 \nof dimensions in a plot was a good representation of our data (Clarke 1993). The NMDS 362 \nplot showed a clear separation of the samples of autoclaved soil in pots where S. 363 \ninaequidens was growing alone (CTR; Fig. 4. Panel A). Samples from the experienced 364 \ncommunity (SA) tended to spread more, while samples from the naïve community (IT) in 365 \nany soil condition clustered more closely (Fig. 4. Panel A). Our PERMANOVA test 366 \naccounted for 47.27% of the overall variation and indicated an effect of both competing 367 \ncommunities and soil, but not their interaction, on ASV community structure 368 \n(PERMANOVA, Community F = 0.23378, p = 0.001, Soil F = 0.11063, p = 0.003; 369 \nSupplementary information, Table S8).     370 \n 371 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n19 \n \n 372 \nFigure 4.  Panel A: Two-dimensional plot of our non-metric multidimensional scaling 373 \nanalysis (NMDS) for bacterial ASVs. The control treatment CTR is shown in light blue, 374 \nthe naïve community from the invasive range IT in purple, and the experienced 375 \ncommunity in the native range SA in turquoise. Autoclaved soil treatment is represented 376 \nwith filled circles (st) and wild soil (not autoclaved) with filled triangles (w). Panel B: 377 \nDifferences among community and soil treatments in ASV richness and Shannon index 378 \nof soil bacterial communities. Autoclaved soil is represented in light pink and wild soil 379 \n(not autoclaved) in green. ASV richness: Interaction, p < 0.05; Shannon index (ANOVA, 380 \nCommunity F = 5.699, p < 0.05, Soil F = 24.082, p < 0.001, Interaction F = 4.254, p = 381 \n0.04). 382 \n 383 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n20 \n \nWe found that the highest richness and diversity (in terms of Shannon index) of ASVs 384 \noccurred in wild soils from the South African communities and the lowest in the 385 \nautoclaved soil with no competing species (control). There was a significant interaction 386 \nbetween competing community types and soil conditions for bacterial ASVs (GLM, p < 387 \n0.05, ANOVA, p < 0.05; Supplementary information, Table S9). Specifically, the effect 388 \nthat community had on ASV richness was modified by soil conditions, with less ASV 389 \nrichness and diversity in controls and South African communities growing in autoclaved 390 \nsoil, compared to wild conditions. Furthermore, ASV richness and diversity in Italian 391 \ncommunities did not differ between soil conditions (Fig. 4. Panel B).  392 \nThere was a statistically significant relationship between both ASV richness and 393 \nShannon index and the height and lateral growth of S. inaequidens (LM height: Adj-394 \nR2(ASV richness) = 0.32, p < 0.01, Adj-R2(ASV Shannon) = 0.18, p = 0.04, LM lateral 395 \ngrowth: Adj-R2(ASV richness) = 0.39, p < 0.01, Adj-R2 (ASV Shannon) = 0.14, p = 396 \n0.067; Fig. 5, Supplementary information, Table S10). Specifically, S. inaequidens 397 \nindividuals were taller and wider when growing in pots with lower soil bacterial diversity.     398 \n 399 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n21 \n \n400 \nFigure 5. Linear model results for the relationship between soil bacterial diversity, 401 \nrepresented as ASV richness and Shannon index, and height and lateral growth of 402 \nSenecio inaequidens (LM height: Adj-R2(ASV richness) = 0.32, p < 0.01, Adj-R2(ASV 403 \nShannon) = 0.18, p = 0.04, LM lateral growth: Adj-R2(ASV richness) = 0.39, p < 0.01, 404 \nAdj-R2(ASV Shannon) = 0.14, p = 0.067). For reference, the identity of the competing 405 \ncommunities is indicated in different colors. 406 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n22 \n \nDiscussion 407 \nWith our experiment, we investigated how the eco-evolutionary experience of species in 408 \nthe resident community and the soil biotic conditions influence the performance of 409 \nSenecio inaequidens. As hypothesized, the identity of competing plant communities 410 \nsignificantly affected S. inaequidens growth, supporting the hypothesis that competition 411 \nwith naïve species in the invasive range is less intense than with experienced species 412 \nfrom the native range. We only found partial support for our other hypotheses. 413 \nSpecifically, the effect of phylogenetic relatedness in explaining S. inaequidens 414 \nperformance was mixed, with no effect of community-wide distances but a significant 415 \ninfluence of the most abundant species relatedness to S. inaequidens. Although the 416 \ncompetitive responses of S. inaequidens to the plant communities were not significantly 417 \naffected by autoclaving the soil in which they grew, soil bacterial diversity still seems to 418 \nplay a role in its performance. 419 \nEco-evolutionary experience and species identity modulates competition 420 \nOur results align with previous studies suggesting that naïve species in the invasive 421 \nrange may lack evolved resistance or competitive strategies against introduced species 422 \nwith which they have had no similar interactions in their evolutionary history (Callaway et 423 \nal. 2011; Saul et al. 2013; Zhang et al. 2018). For instance, in a removal experiment, 424 \nCallaway et al. (2011) found Centaurea stoebe L. populations in their native range 425 \n(Europe) to exhibit a significantly higher response (6.5- to 7.5-fold) to the removal of 426 \nneighboring plants compared to populations in their invasive range (North America). The 427 \nreduced competitive effects associated with the lack of eco-evolutionary experience of 428 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n23 \n \nthe Italian communities could be attributed to several mechanisms, including differences 429 \nin resource acquisition with naïve native competitors. On the other hand, S. inaequidens 430 \nmay be exerting a stronger competitive response, possibly through allelopathic effects 431 \nthat naïve species have not yet adapted to counter. Additionally, Senecio inaequidens is 432 \nknown to contain secondary metabolites in its tissues (i.e. pyrrolizidine alkaloids) that 433 \nare poisonous to some animals (Dimande et al. 2007). Invasive populations may benefit 434 \nfrom this chemical defense, as naïve herbivores in the newly colonized environment are 435 \nunlikely to feed on it, further enhancing its invasion success (Scherber et al. 2003; 436 \nMisuri et al. 2020). Alternatively, its success could be linked to a subtle temporal 437 \nadvantage, allowing it to grow slightly faster and establish dominance earlier in the 438 \ncompetition. Indeed, Delory et al. (2019) found S. inaequidens to exhibit strong 439 \ncompetitive effects on native plants when it has a temporal advantage due to, for 440 \nexample, the slower growth of competing native species (Delory et al. 2019).  441 \nWe also found that the identity of the species in the community affected S. inaequidens 442 \nperformance. In particular, the South African Wahlenbergia androsacea had a 443 \nconsistent negative effect on S. inaequidens height when present in the community. 444 \nThis pattern was not consistent across species from the native range, indicating that 445 \norigin or co-occurrence per se is not a strong indicator of competitive effects of the 446 \nnative species. Instead, the traits of the competing species may play a more significant 447 \nrole. For example, a study modeling experimentally derived competitive impact and 448 \nresponses of Acroptilon repens, a species native to Uzbekistan and invasive in North 449 \nAmerica, found them to be rather dependent on the traits of the species it was 450 \ncompeting with (Xiao et al. 2013). These results together support the idea that invasion 451 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n24 \n \nsuccess and impact are shaped by both the introduced species traits and the 452 \nadaptability and competitiveness of the recipient community.  453 \nOur phylogenetic analyses support the idea that the presence of a dominant, closely 454 \nrelated native species (e.g., Wahlenbergia sp.) may increase the competitive resistance 455 \nagainst S. inaequidens, supporting Darwin’s naturalization hypothesis. This finding is in 456 \nline with the assumption that phylogenetic relatedness can be a good proxy for 457 \nfunctional trait similarity and resource use overlap, leading to more intense competition 458 \n(Divíšek et al. 2018). Our findings also align with previous research showing that biotic 459 \nresistance in native plant communities against other invasive Asteraceae species in 460 \nEurope, such as Ambrosia artemisiifolia L. and Solidago gigantea Aiton, is strongly 461 \ninfluenced by phylogenetic proximity to dominant native species (Yannelli et al. 2017). 462 \nTherefore, while community phylogenetic similarity may not strongly predict invasion 463 \nsuccess (Dostál 2011), interactions with key species within the community, particularly 464 \nthe most abundant ones, may play a critical role. Interestingly, a recent observational 465 \nstudy carried out in Northern Italy described a negative relationship between S. 466 \ninaequidens performance and phylogenetic similarity to resident species in the field 467 \n(Quaglini et al. 2025), lending support to what is known as the pre-adaptation 468 \nhypothesis. The study found that S. inaequidens performed better when growing 469 \nalongside more similar species, particularly in more productive habitats. Such 470 \napparently contradictory results could be reconciled by recent reviews suggesting that 471 \nDarwin’s naturalization and pre-adaptation hypotheses are not mutually exclusive, but 472 \nmay operate at different spatial scales (Thuillier et al. 2010; Ma et al. 2016). Namely, 473 \nsuccessful alien species would be more closely related to natives at broader spatial 474 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n25 \n \nscales, due to environmental filtering, but more distantly related at finer spatial scales, 475 \nwhere competition for limiting resources becomes more important (Ma et al. 2016). In 476 \nother words, at large scales, environmental filtering selects for invaders that are adapted 477 \nto the conditions of the new area, while at small spatial scales, the role of competition 478 \nfor limiting resources becomes more important. This highlights the context dependency 479 \nof biotic resistance, where competition dynamics can shift depending on environmental 480 \nconditions and resource levels. 481 \nReduced soil bacterial diversity benefits Senecio performance under competition 482 \nWe observed distinct proportions of the most abundant bacterial phyla across 483 \ntreatments, mainly Pseudomonadota and Actinomycetota, with South African soils 484 \nexhibiting slightly higher levels of Actinomycetota, while Italian soils had more 485 \nPseudomonadota. Actinomycetota, a highly diverse and globally widespread bacterial 486 \nphylum (van Bergeijk et al. 2020), along with Pseudomonadota, is commonly found 487 \nacross various habitats in Europe (Labouyrie et al. 2023). Autoclaved soil showed a 488 \nsignificantly lowered bacterial diversity compared to wild soil, at least in the control and 489 \nSouth African communities. In those conditions, bacterial communities could not recover 490 \ntheir original diversity after the sterilizing treatment which eliminated to some degree the 491 \nexisting soil microbial community. The composition of the plant community competing 492 \nwith S. inaequidens also affected soil microbial diversity, with the South African 493 \ncommunities supporting higher bacterial diversity in wild soil compared to other 494 \ntreatments. Control pots with S. inaequidens individuals growing alone maintained the 495 \nmost unique bacterial communities, especially in autoclaved soils, whereas soils with 496 \ncompeting native plants showed greater similarity in community structure. For instance, 497 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n26 \n \ncontrol pots with autoclaved soil were characterized by a generally higher abundance of 498 \nNocardioides (10.0% on average) compared to the other treatments. Such results 499 \nsuggest that plant community identity influences microbial assemblages, even after a 500 \nsterilization treatment. One possible explanation for the observed patterns is that 501 \nintroduced plants like S. inaequidens may bring along their associated bacteria (e.g. in 502 \nthe seeds), which can aid their invasion by enhancing establishment, nutrient 503 \nacquisition, growth, or resistance to local biotic pressures (van der Putten et al. 2007; Le 504 \nRoux et al. 2017; Zhang et al. 2023).  505 \nSoil autoclaving did not have a significant direct effect on the overall competitive 506 \nresponse of S. inaequidens to competition, rather bacterial diversity was found to 507 \ninfluence its performance. Autoclaved soils generally supported reduced bacterial 508 \ndiversity, and lower bacterial diversity was associated with increased S. inaequidens 509 \nheight. This finding is somewhat unexpected, given that higher microbial diversity is 510 \ntypically associated with ecosystem stability and resilience (Ehrenfeld 2003; Wardle et 511 \nal. 2004). One possible explanation, consistent with our soil autoclaving results, is that 512 \nreduced microbial diversity may lower the presence or activity of pathogens and 513 \ncompetitors, thereby enabling S. inaequidens to allocate more resources toward growth. 514 \nThis aligns with the enemy release hypothesis, which posits that invasive species may 515 \nescape their natural enemies in new environments, reducing their biotic resistance and 516 \nenhancing their performance (Keane and Crawley 2002; Heger et al. 2024). The 517 \nenormous diversity of soil microbial communities can harbor generalist pathogens that 518 \naffect invasive plants but also disadvantage native species through pathogen spillover, 519 \nespecially if exotics are more tolerant (van der Putten et al. 2007; Dawson and Schrama 520 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n27 \n \n2016). Therefore, another possible explanation for the increased height of S. 521 \ninaequidens in soils with low bacterial diversity is that native plants may be less vigorous 522 \nor competitive under these conditions, possibly due to a shortage of beneficial microbes 523 \nor disruptions of commensalistic and symbiotic relationships between the soil microbial 524 \ncommunity and the plants. With less competition from native plants, S. inaequidens 525 \ncould allocate more resources to growth, leading to taller individuals. This is supported 526 \nby other research showing correlations between S. inaequidens traits, particularly those 527 \nrelated to competitive ability and resource allocation, and bacterial diversity (e.g. 528 \nThébault et al. 2010). These findings suggest that shifts in soil microbial diversity could 529 \ninfluence S. inaequidens ability to outcompete native species, potentially by altering 530 \nnutrient availability, pathogen pressure, or the presence of beneficial microbial partners. 531 \nIt is important to note that methods like autoclaving can alter soil chemistry, nutrient 532 \navailability, and physical structure, potentially confounding experimental results by 533 \naffecting both microbial communities and abiotic factors (Perkins et al. 2013). We note 534 \nthat sterilization does not fully eliminate bacterial DNA, however, its influence is likely 535 \nminimal, as samples were collected when community shifts dominate and residual DNA 536 \nfrom cells killed ~90 days earlier is probably negligible. Finally, the 84-day duration of 537 \nthe experiment provided valuable insights, though longer-term studies could offer a 538 \nmore comprehensive understanding of plant-soil feedbacks and competitive dynamics 539 \n(Liu et al. 2024).  540 \n 541 \n 542 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n28 \n \nConclusions and implications for management 543 \nOur study highlights the interplay between eco-evolutionary experience, plant 544 \nphylogenetic relationships and soil biotic conditions. By analysing the interactions 545 \nbetween this invasive species and plant communities from both its native and invasive 546 \nranges, we provide insights into the possible mechanisms driving its invasion success, 547 \nwhich seems to be favoured by the inexperience of the community of the invasive range 548 \nwith respect to the invader (i.e. naivety). Based on our findings, we argue that selecting 549 \nfew phylogenetically related species at high abundances to outcompete S. inaequidens 550 \ncould be a promising practice for management in areas under restoration. In particular, 551 \nthe observation that S. inaequidens performs better in the presence of naïve species 552 \nand lower microbial diversity indicates that restoration efforts might benefit from 553 \nenhancing the competitive ability of native species and promoting microbial diversity. 554 \nThis could involve the selection of native species that are closely related to the invader 555 \nor have strong competitive abilities and testing soil amendments to increase microbial 556 \ndiversity and resilience. Furthermore, our findings suggest that management strategies 557 \nshould also consider the composition and functional roles of native communities by 558 \nselecting multi-species suites of closely related competitors displaying similar trait 559 \nprofiles, as well as the structure of soil microbial communities.  560 \nAcknowledgements 561 \nFAY acknowledges funding from the Feodor Lynen Fellowship, awarded by the 562 \nAlexander von Humboldt Foundation, and the Rising Star Fellowship, granted by the 563 \nDepartment of Biology, Chemistry, and Pharmacy at Freie Universität Berlin. 564 \nAuthor-formatted, not peer-reviewed document posted on 03/06/2025. DOI:  https://doi.org/10.3897/arphapreprints.e160941\n\n29 \n \nReferences 565 \nAbbott KC, Karst J, Biederman LA, Borrett SR, Hastings A, Walsh V, Bever JD (2015) 566 \nSpatial Heterogeneity in Soil Microbes Alters Outcomes of Plant Competition. 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