Above- and below-ground mechanisms enhance competitiveness of invasive Phytolacca americana in heavy metal-rich soils

Increasing evidence suggests that invasive plants can sustain high growth rates in heavy metal-enriched environments, while native species do not contribute to their invasion success. However, the underlying mechanisms are poorly understood. This study examined the invasive plant Phytolacca americana and native Phytolacca acinosa in Cd-contaminated soils in China. The results showed that, while native plant biomass decreased at higher soil Cd concentrations, invasive plant biomass remained unaffected. Soil enzyme activities decreased with increasing Cd concentration but were higher in soils where invasive plants grew than those in soils where native plants grew. Phytolacca americana accumulated more Cd, particularly in the leaves and under higher soil Cd concentrations. Native seed germination was inhibited with increasing litter-Cd concentration, whereas invasive seed germination was unaffected. These findings suggest that the ability of the invasive species to mitigate the negative effects of Cd pollution on soil enzyme activities contributed to their higher Cd-tolerance by maintaining soil nutrient availability. Additionally, higher leaf-Cd levels in invasive plants constitute an elemental defence. Overall, heavy metal pollution not only favoured invasive plants by suppressing native plant growth but may have also promoted invasion through the effects of Cd accumulation in invasive litter on native seed germination. Above- and below-ground mechanisms enhance competitiveness of invasive Phytolacca americana in heavy metal-rich soils Zhisen Yan 1,5, Yuxin Lai 1,5, Jingru Zhang 2, Shaoyu Zhang 1, Yunshan Liu 1, Bo Li 1, Evan Siemann 4, Jihua Wu 3* and Yi Wang 1* 1 State Key Laboratory for Vegetation Structure, Functions and Construction, Ministry of Education Key Laboratory for Transboundary Ecosecurity of Southwest China, and Yunnan Key Laboratory of Plant Reproductive Adaptation and Evolutionary Ecology, Institute of Biodiversity, School of Ecology and Environmental Science, Yunnan University, 650500, Kunming, China 2 Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, National Observations and Research Station of Wetland Ecosystems of the Yangtze Estuary, Institute of Biodiversity Science and Institute of Eco-Chongming, School of Life Sciences, Fudan University, Shanghai, China 3 State Key Laboratory of Herbage Improvement and Grassland Agro-Ecosystems, College of Ecology, Lanzhou, University, Lanzhou, China 4 Department of Biosciences, Rice University, Houston, TX 77005, USA 5 Equal Contribution Zhisen Yan Email: [email protected] Yuxin Lai Email: [email protected] Jingru Zhang Email: [email protected] Shaoyu Zhang Email: [email protected] Yunshan Liu Email: [email protected] Bo Li Email: [email protected] Evan Siemann Email: [email protected] * Correspondence: Jihua Wu Email: [email protected] Yi Wang Email: [email protected]

Increasing evidence suggests that invasive plants can sustain high growth rates in heavy metal-enriched environments, while native species do not contribute to their invasion success. However, the underlying mechanisms are poorly understood. This study examined the invasive plant Phytolacca americana and native Phytolacca acinosa in Cd-contaminated soils in China. The results showed that, while native plant biomass decreased at higher soil Cd concentrations, invasive plant biomass remained unaffected. Soil enzyme activities decreased with increasing Cd concentration but were higher in soils where invasive plants grew than those in soils where native plants grew. Phytolacca americana accumulated more Cd, particularly in the leaves and under higher soil Cd concentrations. Native seed germination was inhibited with increasing litter-Cd concentration, whereas invasive seed germination was unaffected. These findings suggest that the ability of the invasive species to mitigate the negative effects of Cd pollution on soil enzyme activities contributed to their higher Cd-tolerance by maintaining soil nutrient availability. Additionally, higher leaf-Cd levels in invasive plants constitute an elemental defence. Overall, heavy metal pollution not only favoured invasive plants by suppressing native plant growth but may have also promoted invasion through the effects of Cd accumulation in invasive litter on native seed germination. Keywords: Aboveground and belowground, Heavy metals, leaf litter, Phytolacca americana, Phytolacca acinose, seed germination 1 Introduction As countries worldwide become increasingly interconnected, and the number of invasive species continues to rise (Seebens et al., 2021), invasive alien species have become a global ecological-security issue, seriously threatening the sustainable development of human society and biodiversity (Courchamp et al., 2017; Pimentel et al., 2001; Pysek et al., 2017). In addition, owing to accelerated industrialisation, environmental pollution has worsened with heavy-metal pollution, which has a particularly extensive and profound impacts on ecosystems (Ashraf et al., 2019; Ha et al., 2014). Alien species show high environmental adaptability and epigenetic plasticity, rapidly adapting to biotic and abiotic stress conditions; hence, they tend to readily outcompete native species (Davidson et al., 2011; Jardeleza et al., 2021; Liu et al., 2023). Environmental pollution can exacerbate current biological invasions and facilitate invasion by alien species (Sun et al., 2023) depending on its relative impact on alien and native species (Gioria et al., 2023b). Habitats with severe environmental pollution may show low levels of native biodiversity and empty ecological niches, and are more vulnerable to invasion by alien species (Pautasso et al., 2010). In addition, alien plant species often show rapid growth, high reproduction rates, and efficient resource use, and may become locally more abundant if they are relatively more tolerant to environmental pollution (Bradley et al., 2010; Yang et al., 2007). Moreover, many invasive species not only tolerate (Prabakaran et al., 2019; Yuan et al., 2016) but also hyperaccumulate (Peng et al., 2008) and use high concentrations of heavy metals to enhance their competitive advantage over native species through two mechanisms. Heavy metal accumulation in leaves can increase plant resistance to disease and herbivory, a phenomenon known as elemental defence (Fones et al., 2010; Fones and Preston, 2013; Reeves et al., 1981). This mechanism has been shown in invasive plants such as Ageratina adenophora which accumulates Cd in its leaves, thereby reducing damage caused by Rhizoctonia solani (Dai et al., 2020); however, it is not clear whether differences in elemental defence contribute to invasion (Wang et al., 2022). As for the second mechanism, heavy metal accumulation may affect the growth of neighbouring plants by altering the content, distribution, and available forms of heavy metals in the surrounding surface soil; a phenomenon known as elemental allelopathy (Morris et al., 2008). Compared to native plants, invasive plants may show higher productivity, stronger heavy metal-accumulation capacity, and faster rates of litter decomposition (Allison and Vitousek, 2004; McLeod et al., 2016). Therefore, they may release larger amounts of heavy metals into the surrounding topsoil layer, affecting seed germination, seedling survival, and soil microbial communities (Gall et al., 2015). If the impacts of heavy metals on native seeds are greater than those on the seeds of invasive plants, such heavy metal enrichment via litter may accelerate the displacement of native plants. Recently, there has been considerable scientific interest in the mechanisms by which invasive plants enhance their competitiveness in heavy metal-contaminated soils. Because the most successful invasions by alien plants are driven by a combination of above and belowground processes (Gioria et al., 2023a), the exploration of these mechanisms should not be limited to the aboveground parts of the plant body, but should consider belowground plant organs as well. For instance, heavy metals in soils can reduce the activity of enzymes that are critical for the breakdown of organic compounds that provide nutrients to plants (Yeboah et al., 2021). Because invasive plants often stimulate higher levels of soil enzyme activity than native plants (Zhang et al., 2019), they may be able to alleviate the negative effects of heavy metals on soil enzyme activity. Further, heavy metals accumulated in the tissues of invasive plants can help increase their resistance to natural enemies. Nonetheless, the mechanisms by which invasive species use a combination of above and belowground mechanisms involving heavy metals to extend their competitive advantage over native species and enhance their invasiveness remains unclear. Phytolacca americana (Phytolaccaceae) was introduced to China in 1935 and has become invasive in natural ecosystems, including agricultural and forestry lands, in more than 20 provinces in China (Xu et al., 2012). Further, the species shows greater tolerance to herbivore attack in the presence of Mn pollution (Zhou et al., 2023), accumulates Mn and Cd (Peng et al., 2008), and stimulates Lactobacillus bacteria, which may influence Cd bioavailability (Li et al., 2023). It is not associated with Mn- or Cd-rich soils in its native habitat and is thought to hyperaccumulate such metals owing to natural selection for soil acidification, which increases P availability (DeGroote et al., 2017). The native species, P. acinose, co-occurs with P. americana in China and has similar characteristics. It is a traditional medicinal plant with a 2000-year history of medicinal use. Because of the P. americana invasion, P. acinosa has become rare, affecting the P. acinosa collection and processing industry in Yunnan Province (Liu et al., 2022; Xiao et al., 2022). Here, we used the heavy metal hyper-accumulating invasive plant, Phytolacca americana, and its native congener (Phytolacca acinosa) to test the following predictions in an experiment conducted under soil Cd-addition treatment in China: (1) In Cd-contaminated soils, t he invasive plant maintains higher levels of soil enzyme activity and accumulates higher levels of Cd in its tissues, and sustains higher growth rates compared to the native plant; (2) In Cd-contaminated soils, the invasive plant will produce larger amounts of Cd litter, which will have a more negative impact on the germination of native seeds than that of its own seeds. not-yet-known not-yet-known not-yet-known unknown 1 2 Materials and Methods 1.1 2.1 Experimental plant material In July 2022, we collected mature fruits of P. americana and P. acinosa from wild populations across multiple sites in Kunming (Chenggong District) and Qujing, Yunnan Province. All sampling locations were subjected to minimal human disturbance. A minimum distance of 2 km was used to separate the sampled populations. Five to ten plants were selected at random, and seeds were harvested by removing the pulp and allowing them to air-dry. Subsequently, seeds from each species were randomly selected for inclusion in the experiments (Xiao et al., 2022). 1.2 2.2 Soil treatment In April 2023, soil samples were collected from a typical broad-leaved forest in Kunming (Chenggong District). Samples were collected from five locations with no history of Cd pollution or occupation by either of the species used for the experiments. Leaf litter was removed from the ground surface and the soil samples were collected to a depth of 40 cm, air-dried, and sieved through a 0.9 mm mesh to remove impurities, animals, and plant roots. We filled 144 10-L pots (22 cm top diameter, 12.5 cm bottom diameter, 40 cm high) with soil. Each pot was assigned to a plant (invasive, native, or plant-free control) and a Cd concentration (0, 4, 16, or 64 mg/kg of dry soil) treatment in a factorial design. These concentrations were similar to those reported for polluted soils near mining operations (Kabir et al., 2012). Pots were supplied with 500 ml of cadmium chloride (CdCl2) solution (2, 8, or 32 mg Cd/kg dry soil). After two months, the Cd treatment was repeated. An equal volume of water was added to the control groups. 1.3 2.3 Plants In May 2023, we soaked the seeds in concentrated sulfuric acid to break dormancy and washed them with deionised (DI) water. Floating seeds were discarded and the remaining seeds were placed in 1% agar medium. Once the radicles of the seeds on the medium grew to approximately 1 cm, the germinated seeds were transferred to a plastic box containing soil (without Cd addition) and continued under culture in an incubator (relative humidity 70%, light/dark cycle of 16/8 hours, temperatures of 21/16℃). Approximately 14 days later, when the seedlings had grown two or three leaves, seedlings of equivalent size were randomly selected and transplanted into pots in the greenhouse located at Yunnan University (24°50’ N, 102°52’ E) in Kunming (Chenggong District). The daytime temperature within the greenhouse was maintained at 30℃ and the night-time temperature at 18℃, with relative humidity ranging between 50%-80%. Seedlings that died within two weeks of transplanting were immediately replaced. Seedlings of other species that germinated in the pots were regularly removed. The positions of the pots were randomised and changed monthly, and the plants were watered with 2500 ml at two-day intervals. 1.4 2.4 Plant harvesting and measurements After 120 days of growth (August 2023), plants were clipped at ground level, stems and leaves were separated, and the soil was shaken from the roots prior to thorough mixing and passing through a 40-mesh sieve. Sieved samples were then placed in centrifuge tubes and stored at 4℃ for subsequent analytical testing. Then, roots were washed. In turn, plant samples were oven-dried for 72 h at 60℃ and weighed. Dried leaf and root samples were placed separately in a mortar and ground to a powder that was then passed through an 80-mesh sieve and stored in centrifuge tubes for subsequent testing. The heavy metal contents in the leaves, roots, and soil were measured using inductively coupled plasma mass spectrometry (PE2000 ICP-MS system, Perkin Elmer Waltham, MA, USA). Soil enzyme activities including, urease, acid phosphatase, and dehydrogenase, were measured using commercial kits (Jiangsu Addison Biotechnology Co., Ltd.), according to manufacturer instructions. 1.5 2.5 Leaf litter collection We set up 96 additional pots in the greenhouse and assigned them to plant (invasive or native) and Cd (0, 4, 16, or 64 mg/kg) treatments, following the methods described above. Leaves were harvested at the end of the growing season (approximately 6 months) when their colour started to fade from green to yellow. Leaves were air-dried in the laboratory and used for seed germination and decomposition experiments. 1.6 2.6 Seed germination experiment One-hundred and eighty plastic boxes (17.5 cm×12 cm×6.5 cm) were filled with soil from the same field. Then, 10 seeds of the invasive and native species were placed separately on the soil surface. We added 0.5 g of litter collected from one of the pots for each litter species and Cd addition combination treatments to ten boxes of each species. The remaining ten boxes of each species did not receive any litter (i.e. litter-free controls). Therefore, the design was as follows: two germinating species × (control [no litter] + [four Cd treatments (0, 4, 16, and 64 mg/kg] × 2 litter species)) × 10 replicates = 180 boxes. Every 24 h, 50 ml of DI water was uniformly sprayed onto the soil surface in each box. The boxes were placed in a light incubator for continued observation (relative humidity 70%, light/dark cycle of 16/8 hours, day/night temperatures of 21/16℃); seed germination was recorded at three-day intervals. 1.7 2.7 Leaf litter-decomposition experiment One gram of leaf litter (from a single pot) was placed in nylon mesh bags (mesh size: 40). Leaf litter was folded instead of cut to pieces, such that decomposition would better mirror natural processes (Cheng et al., 2022). Then, the bags were individually placed in square pots containing 15 g of field soil (10 × 10 × 7.5 cm) and covered with a thin layer of soil. The pots were kept in a greenhouse under consistent environmental conditions (daytime average temperature of 30°C, night-time average temperature of 18°C, relative humidity of 50%-80%). Soil moisture content was maintained at 60%. Litter-containing nylon bags were collected and brought back to the laboratory after one, three, or five months. The remaining leaf litter in the bags was washed with DI water over a 0.1 mm screen to remove adhering soil, and then dried to constant weight at 60℃. Soil Cd was measured before and after five months of litter decomposition. 1.8 2.8 Statistical analysis Analysis of variance (ANOVA) was used to test whether aboveground mass, belowground mass, root to shoot ratio (R:S), and total mass depended on plant species, Cd addition rate (a 4-level categorical variable), and their interactions as fixed effects. We used adjusted-means contrast tests to distinguish among means for significant results in these and other ANOVA tests. Additionally, we used repeated-measures ANOVA tests to study how Cd concentrations in plant tissues depended on plant species, tissue type, Cd addition (4-level categorical variable), and their interactions as fixed effects. Repeated-measures ANOVA was also used to control for the non-independence of leaf and root measurements from the same plant. We used ANOVA to test whether Cd, urease, dehydrogenase, or acid phosphatase depended on plant treatment (3-level variable), Cd addition rate (4-level categorical variable), and their interactions as fixed effects. To investigate any differences in germination rates due to differences in litter type independent of Cd concentration, we performed an ANOVA using only water-free controls and litter from pots without Cd addition. This ANOVA test used germination species, litter treatment, and their interaction, and fixed terms, and the log of the odds of seed germination (ln [germinating/not germinating]) as the response variable. To investigate any differences in the responses of P. americana and P. acinosa seeds to Cd concentrations in the litter, we first estimated the Cd concentrations in the added litter using the average values measured in the first experiment. We then performed a mixed model regression with the log odds of germination as the response variable and estimated Cd concentration as the predictor and fitted different slopes and intercepts for P. americana vs. P. acinosa seeds. A slope-contrast test was performed to determine whether the slopes differed. Additionally, we used ANOVA to test the dependence of the leaf litter decomposition rate (-ln[remaining mass/starting mass]) on the fixed effects of species, Cd treatment, decomposition time, and their interactions, along with a random term for pot (in which litter was produced) nested in species × Cd addition. Lastly, we used ANOVA to test the dependence of soil Cd concentration on species, Cd addition treatment, and their interaction, after five months of litter decomposition. All analyses were performed using SAS, version 9.4. 1 2 Materials and Methods not-yet-known not-yet-known not-yet-known unknown 1 3 Results 1.1 3.1 Effects of Cd on the growth of the native and invasive plants Aboveground mass accumulation depended on the interaction between species and Cd concentration (Supplementary Figure S1”). Larger mass accumulation was observed in plants of P. americana along with positive effects of increasing Cd concentrations on P. americana and negative effects on P. acinosa (Table 1, Figure 1a). Concomitantly, belowground mass decreased with increasing Cd concentration and was lower in P. acinosa (Figure 1b, Table 1). Therefore, the R:S ratio decreased with increasing Cd concentration (Figure 1c). In turn, total mass of P. americana was higher than that of P. acinosa and did not vary with Cd concentration, whereas that of the latter decreased with increasing Cd concentration (Figure 1d, Table 1). 1.2 3.2 Cd accumulation and transport ability of the native and the invasive plant species The amount of Cd in the plant tissues depended on the interaction between species, Cd addition treatment, and plant part (Table 2). In general, higher Cd levels were observed in the tissues of P. americana than in those of P. acinosa, particularly in the tissues of plants grown in soils under higher Cd treatments, and differences between root and leaf Cd concentrations in some species and Cd treatments were significant (Figure 2). Specifically, P. americana had higher Cd concentrations in the roots of plants Cd-untreated plants, but higher Cd concentrations in the leaves across Cd treatments. Furthermore, P. acinosa showed higher Cd concentrations in the leaves of plants grown under 16 mg Cd (Figure 2). The differences in Cd concentrations in the leaves were largest under the 64 mg Cd, and those in P. americana were 2.6 times higher than those observed in P. acinosa . 1.3 3.3 Effects of the invasive vs. native plants on Cd remaining in the soil The Cd concentration remaining in the potted soil at the end of the experimental period depended on the interaction between plant species and Cd rate (Table 3). The leaf Cd concentrations were similar in the plant-free control pots, i.e. Cd-untreated (Figure 3). Further, the amount of Cd in the soil increased with increasing Cd rate, and the concentration was consistently lower in plant tissues than in the plant-free control pots (Figure 3). Lastly, Cd levels were lower in P. americana than in P. acinosa soils at the higher Cd concentrations (16 or 64 mg/kg; Figure 3). 3.4 Effects of Cd on soil enzyme activities in the presence of the native vs. invasive plant species Soil urease and acid phosphatase activities depended on plant type and Cd treatment but not on their interaction (Table 3). The results indicated that the P. americana pots showed the highest urease and acid phosphatase activities, whereas no-plant pots had the lowest, and P. acinosa pots showed intermediate enzyme activity levels together with lower urease and acid phosphatase activities with increasing Cd treatment (Figure 4a, c). Soil dehydrogenase activity was dependent on the interaction between plant type and Cd treatment (Table 3). These findings indicate that P. americana pots showed the highest urease activity, no-plant pots had the lowest urease activity, and P. acinosa pots showed intermediate activities, together with lower urease activity under increasing Cd concentration, only in plant-bearing pots (Figure 4b). 1.4 3.5 Effects of Cd-enriched leaf litter input on seed germination For litter from Cd-untreated pots and litter-free controls, germination was higher for P. americana vs. P. acinosa seeds (8.5 vs. 5.9 per 10 seeds, F1,54 = 44.0, P <0.0001). On average, germination was lowest for P. americana litter (5.5/10), highest for no-litter controls (8.6/10), and intermediate for P. acinosa litter (7.5/10) (F2.54 = 21.1, P germinating species and litter addition (F2,54 = 0.3, P = 0.8096). Germination odds for P. americana seeds did not depend on the Cd estimated concentrations in the added litter (F1,154 = 0.4, P = 0.5332), but P. acinosa seeds showed reduced germination with increasing Cd concentration (F1,154 = 9.2, P = 0.0029). Slope contrasts showed that these patterns were significantly different (F1,154 = 6.3, P = 0.0134) (Figure 5). 1.5 3.6 Leaf litter decomposition and its effects on the soil The rate of litter-mass loss depended on litter species, Cd treatment, and decomposition time but not on their interactions (Table 4). The litter of P. americana was decomposed more rapidly than that of P. acinosa (Figure 6a). Litter decomposition rate was faster for litter produced in Cd-untreated than in Cd-treated soils, although all Cd-treated soils showed similar litter mass-loss rates. Further, after five months of decomposition, soil Cd concentrations depended on the interaction between species and Cd treatment (species: F1,88 = 250.2, P <0.0001; Cd: F3,88 = 274.0, P <0.0001; sp × Cd: F3,88 = 74.2, P <0.0001). This finding indicates that greater concentrations of Cd accumulated in the soil when litter was produced in soils treated with higher Cd concentrations, especially in pots in which the invasive species was grown (Figure 6b). 1 3 Results not-yet-known not-yet-known not-yet-known unknown 1 4 Discussion In this study, we investigated how invasive plants enhance their competitiveness over that which characterises native plants and, consequently, their invasiveness, by accumulating heavy metals absorbed by the roots, transferring them to aboveground tissues, and then returning them to the surface soil through the decomposition of leaf litter inputs. Our findings demonstrate that the invasive species analysed herein uses a combination of above- and belowground mechanisms to improve their competitiveness against native plants. Our results revealed significant differences between invasive and native plants in their ability to tolerate, accumulate, and transfer heavy metals when grown in heavy metal-contaminated soils, which in turn have distinct effects on soil enzyme activities. Additionally, we found that the leaf litter of invasive plants containing high Cd concentrations significantly reduced the seed germination rate of native plants by increasing the concentration of Cd in the surface soil. 1.1 4.1 Differences in tolerance, bioaccumulation, and transfer of Cd between invasive plants and native plants Heavy metals are one of the most important pollutants, and their toxicity poses both ecological and environmental challenges (Nagajyoti et al., 2010); yet the emission of heavy metal pollutants continues (Zhou et al., 2020). Although heavy metals can be toxic to plants (Nagajyoti et al., 2010), invasive plants are reportedly more tolerant to heavy metal pollution than native plants, such that heavy metal pollution can in fact facilitate invasion (Gulezian et al., 2012; Lin et al., 2024a). Indeed, plants living in extremely stressful environments often exhibit lower growth performance because they must commit more resources to stress tolerance responses (Laitinen and Nikoloski, 2022; Ogawa-Ohnishi et al., 2022). However, the spread of invasive plants is usually not limited by environmental contamination, as invasive species often show higher tolerance and adaptability (Sun et al., 2009), and thus greater competitiveness than native species when growing together under stressful conditions. Our results confirmed that, compared to native P. acinosa, invasive P. americana was more tolerant to Cd stress, with no decrease in growth, even at the highest Cd concentration, suggesting that Cd pollution favoured its invasiveness. In addition, the results showed that the invasive plant P. americana had a stronger enrichment and transfer ability for Cd, and the concentrations of this heavy metal in the leaves and roots were significantly higher than those in its native congener. Similarly, a previous study found that P. americana accumulated higher Mn concentrations than P. acinosa in its foliage, which may play a role in its release from herbivory (Zhou et al., 2023). Similar results have been previously reported suggesting that heavy metals (e.g. Cd) in the tissues of the invasive plant Alternanthera philoxeroides can promote invasion by increasing plant resistance to herbivory over that of its native congener Alternanthera sessilis (Lin et al., 2024b; Wang et al., 2022). 1.2 4.2 Effects of heavy metal enrichment by invasive plants on soil bioactivity Soil biota can have profound effects on alien-plant invasion (Inderjit and van der Putten, 2010). Microorganisms play a crucial role in this process by influencing the growth and development of the species, thereby influencing competition between invasive and native plants (Mariotte et al., 2018). In soil ecosystems, heavy metals generally have toxic effects on soil microorganisms and can adversely affect the structure of microbial communities (Khan et al., 2010; Lorenz et al., 2006). In particular soil enzyme activities are useful indicators while assessing the impact of heavy metals on soil biological functions. Thus, soil enzymes such as urease and dehydrogenase are sensitive to heavy metals, including Cd, and can reflect the toxicity of these metals to soil microorganisms (Moreno et al., 2001). Our findings are consistent with earlier research showing a significant negative effect of Cd on soil enzyme activities (Shen et al., 2005), and that increased heavy-metal concentrations significantly inhibited the activities of urease, dehydrogenase, and acid phosphatase in the soil (Mao et al., 2015; Xin et al., 2017). The toxicological effects of heavy metals on soil enzyme activities, such as the interference with the ability of microbial cells to synthesise enzymes, are significantly influenced by their concentration (Yeboah et al., 2021). In this study, soil enzyme activities in the presence of the invasive species were significantly higher than those of observed in the presence of the native species, presumably owing to their heavy metal greater bioaccumulation-capacity, which reduced the content of heavy metals in the soil. Depending on the effects of higher soil enzyme activities on invasive versus native plants, this may further affect the invasion process. 1.3 4.3 Effect of leaf litter input on plant fitness The seed germination patterns observed herein suggest that both allelopathy and elemental allelopathy potentially affected the interactions between P. americana and P. acinosa . In particular, we observed that, compared to the litter-free controls, the addition of leaf litter produced in control (i.e. no Cd addition) soils reduced the germination rates of seeds of both species, which is consistent with allelopathic effects on seed germination. The reasons behind the suppression of seed germination by leaf litter are complex, potentially involving physical, chemical, and biological factors, or a combination of these (Cavieres et al., 2006) (Yirdaw and Leinonen, 2002). Although P. americana leaf litter had a stronger negative effect than P. acinosa litter, this effect did not differ between the seeds of the two species, suggesting that allelopathic effects on seed germination do not play a strong role in P. americana invasion into areas dominated by P. acinosa . In contrast, the elemental allelopathic effects on seed germination have the potential to promote invasion by P. americana . The invasive plants produced litter with higher concentrations of Cd, which negatively affected the germination of the native plants without affecting the germination of the invasive ones. This may reflect both the higher concentrations of Cd in the litter as well as its faster decomposition. Therefore, invasive plants growing in heavy metal-polluted soils can change the soil nutrient content through aboveground litter decomposition. By introducing heavy metals into the surface soil and inhibiting the germination of native plant seeds, they can enhance their own fitness, maintain a competitive advantage, and promote their spread and dispersion. Similar effects of elemental allelopathy in promoting invasion have been shown for salts, nickel, and zinc (Morris et al., 2008). The allelopathic effects of Cd on the growth (but not germination) of neighbouring plants have been shown in Arabidopsis elleri interacting with plants in their native range (Reeves et al., 2018); however, our study is the first to show that they may play a role in invasion by alien plants. Further, given that more than 700 plant species hyperaccumulate heavy metals (Pollard et al., 2014), heavy metal allelopathy may play a role in many other invasions. 1 4 Discussion 5 Conclusions In this study, we showed that multiple mechanisms involving Cd potentially contribute to P. americana invasion. First, compared with its native congener, P. americana was more tolerant to higher soil Cd concentrations, both in terms of plant growth and seed germination. Secondly, P. americana mitigated the effects of Cd on soil enzyme activities that have the potential to shape plant interactions. Thirdly, higher concentrations of Cd in the soil seemingly facilitated P. americana invasion via elemental allelopathy, whereby the invasive species accumulated Cd, which in turn negatively impacted the germination of the seeds of the native species but not that of its own seeds. Heavy metal pollution and the introduction of alien species are expected to continue, and heavy metal hyperaccumulation is a trait possessed by many plant species. The set of effects reported here for P. americana may explain the invasive success of other species as well.

The study was supported by the National Key Research and Development Program of China (2023YFC2604500 and 2022YFC2601100), the National Natural Science Foundation of China (U2102218 and 32371751) and Scientific Research Fund project of Yunnan Education Department (2023Y0208). Author contributions Zhisen Yan and Yuxin Lai: conceptualization, data curation, formal analysis, investigation, methodology, project administration, validation, visualization, writing—original draft; Jingru Zhang, Shaoyu Zhang, and Yunshan Liu: formal analysis, investigation, methodology; Jihua Wu and Bo Li: data curation, methodology, project administration, resources; Evan Siemann: data curation, formal analysis, methodology, resources, software, writing; Yi Wang: conceptualization, funding acquisition, investigation, project administration, resources, supervision, validation, writing—original draft. | Species | Cd addition | Species × Cd | |||| | Variable | F 1,88 | P | F 3,88 | P | F 3,88 | P | | Aboveground mass | 322.8 | <.0001 | 0.4 | 0.7230 | 6.6 | 0.0004 | | Belowground mass | 102.0 | <.0001 | 4.9 | 0.0033 | 2.0 | 0.1191 | | Total mass | 340.3 | <.0001 | 1.1 | 0.3479 | 6.6 | 0.0004 | | R:S | 0.1 | 0.7637 | 4.1 | 0.0089 | 1.0 | 0.3895 | Table 2 The dependence of Cd concentrations in plant tissues on the fixed effects species, Cd addition treatment, root vs. leaf and their interactions in a repeated measures ANOVA. Cd concentrations were log transformed. Significant results are indicated in bold (P < 0.05). not-yet-known not-yet-known not-yet-known unknown | Effect | df | F | P | | Species | 1,88 | 232.4 | <0.0001 | | Cd addition | 3,88 | 1723.4 | <0.0001 | | Root vs. leaf | 1,88 | 16.9 | <0.0001 | | Species × Cd addition | 3,88 | 10.6 | <0.0001 | | Species × RvsL | 1,88 | 15 | 0.0002 | | Cd addition × RvsL | 3,88 | 9.4 | <0.0001 | | Species × Cd addition × RvsL | 3,88 | 4.5 | 0.0053 | Table 3 The dependence of Cd concentration, urease activity, dehydrogenase activity and acid phosphatase activity in soil on the fixed effects species (no plants, invasive or native), Cd addition treatment, and their interaction in ANOVAs. Cd concentrations were log transformed. Significant results are indicated in bold (P < 0.05). | Plant treatment | Cd addition | Plant × Cd | |||| | Variable | F 2,132 | P | F 3,132 | P | F 6,132 | P | | Cadmium | 43.3 | <0.0001 | 2890.9 | <0.0001 | 3.5 | 0.0030 | | Urease | 607.9 | <0.0001 | 45.5 | <0.0001 | 1.3 | 0.2800 | | Dehydrogenase | 769.3 | <0.0001 | 44.5 | <0.0001 | 10.4 | <0.0001 | | Acid phosphatase | 790.9 | <0.0001 | 99.0 | <0.0001 | 0.7 | 0.6516 | Table 4 The dependence of leaf litter decomposition rate (-ln[remaining mass / starting mass]) on the fixed effects species, Cd addition treatment, decompose time and their interactions along with a random term for pot nested in species × Cd addition in an ANOVA. Significant results are indicated in bold (P < 0.05). | Fixed effects | df | F | P | | Species | 1,88 | 14.0 | 0.0003 | | Cd addition | 3,88 | 5.9 | 0.0011 | | Species × Cd addition | 3,88 | 2.0 | 0.1245 | | Time | 2,176 | 40.1 | <.0001 | | Species × Time | 6,176 | 0.6 | 0.5760 | | Cd addition × Time | 6,176 | 1.5 | 0.1725 | | Species × Cd addition × Time | 6,176 | 0.6 | 0.7340 | | Random effect | z | P | | | Pot (Species × Cd addition) | 0.4 | 0.6871 | not-yet-known not-yet-known not-yet-known unknown Figure 1 Effects of different amounts of soil Cd addition on the biomass of the invasive plant (P. americana ) and the native plant (P. acinosa ). (a) aboveground biomass, (b) belowground biomass, (c) Total biomass, (d) Root-to-shoot ratio, R:S. Data shown are means ± 1 SE. Means with the same capital letter did not differ in post-hoc tests significantly (P > 0.05). not-yet-known not-yet-known not-yet-known unknown Figure 2 Effects of different amounts of Cd added to the soil on tissue Cd concentration of invasive (I = P. americana ) and native (N = P. acinosa ) plants. Data shown are means ± 1 SE. Means with the same capital letter did not differ in post-hoc tests (P > 0.05) significantly. Horizontal dotted lines indicate the concentration of Cd added to the soil. Figure 3 Effects of different amounts of Cd added to the soil on soil Cd concentration at the end of the experiment in pots without a plant (CK) or with an invasive (I = P. americana ) or a native (N = P. acinosa ) plant. Data shown are means ± 1 SE. Means with the same capital letter did not differ in post-hoc tests (P > 0.05) significantly. Horizontal dotted lines indicate the concentration of Cd added to the soil. Figure 4 Effects of different amounts of Cd added to the soil on soil enzyme activities at the end of the experiment in pots without a plant (CK) or with an invasive (I = P. americana ) or a native (N = P. acinosa ) plant. (a) Urease, (b) Dehydrogenase, (c) Acid phosphatase. Data are means ± 1 SE. Means with the same capital letter did not differ in post-hoc tests (P > 0.05) significantly. MTTF = MTT formazan. PNP= p-nitrophenol. Figure 5 The dependence of log odds of germination (back-transformed) of invasive P. americana (blue) vs. native P. acinosa seeds (red) on the estimated concentrations of Cd in litter (square-root transformed). The letters indicate the treatment in which the litter was produced (N0 = native 0 mg Cd / kg, I0 = invasive 0 mg Cd / kg, N4 = native 4 mg Cd / kg, I4 = invasive 4 mg Cd / kg, N16 = native 16 mg Cd / kg, I16 = invasive 16 mg Cd / kg, N64 = native 64 mg Cd / kg, I64 = invasive 64 mg Cd / kg). The solid line indicates a significant slope (P <0.05). Figure 6 (a). The proportion of litter mass of the invasive ( P. americana ) vs. native ( P. acinosa ) species remaining after 1, 3 or 5 months of decomposition for litter produced at various Cd treatment to the soil. (b). The concentration of Cd in surface soil after five months of decomposition with litters of the invasive vs. native species produced under different Cd concentration treatments. Data shown are means ±1 SE. Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 263views 164downloads Citations Download citation Zhisen Yan, Yuxin Lai, Jingru Zhang, et al. Above- and below-ground mechanisms enhance competitiveness of invasive Phytolacca americana in heavy metal-rich soils. Authorea. 08 February 2025. DOI: https://doi.org/10.22541/au.173899094.41796786/v1 DOI: https://doi.org/10.22541/au.173899094.41796786/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu. Cited by - Invasive Plants as Accumulators of Heavy Metals and Potentially Toxic Elements: A Review with Implications for Remediation, Plants, 15, 7, (1078), (2026).https://doi.org/10.3390/plants15071078 Loading...