Introduction

Over thirteen thousand alien plant species have already become naturalized (i.e. established self-sustaining populations outside cultivation) beyond their native ranges (van Kleunen et al. 2015). The number of these naturalized alien plant species continues to rise with no signs of saturation (Seebens et al. 2017), and is projected to increase by about 18% from 2005 to 2050 (Seebens et al. 2021). As naturalization is a major step in the plant invasion process, the increase in the number of naturalized species may also result in more species becoming invasive, and thus having severe environmental, economic and human health impacts (Vilà et al. 2011; Schaffner et al. 2020; Diagne et al. 2021). Concerns over the impacts of plant invasions have stimulated considerable interest in the mechanisms underlying plant invasions (van Kleunen et al. 2010; Funk 2013; Zhang et al. 2020a, b; Liu et al. 2021). However, despite considerable progress in our understanding of invasions, it is still unclear why some plant species, despite similar degrees of commonness in their native range, are more successful as aliens than others.

The fluctuating-resource hypothesis poses that an alien plant species could establish more easily in a community if the availability of a limiting resource (e.g. nitrogen) increases (Davis and Thompson 2000). Several studies found support for this hypothesis, and indicated that many successful alien species are opportunists that capitalize more strongly on additional resources than less successful species (Richards et al. 2006; Dawson et al. 2012b). For example, common alien species showed stronger biomass increases in response to nutrient addition than rare alien species (Dawson et al. 2012a). Similarly, Parepa et al. (2013) found that invasive Fallopia spp. became more dominant after an increase in nutrients. It was also shown that Central European plant species from more productive habitats are more likely to be invasive elsewhere in the world than species from less productive habitats (Dostál et al. 2013). However, although Liu and van Kleunen (2019) showed that Central European plant species that are widely naturalized elsewhere grow faster than the ones that are not widely naturalized, this was not related to differences in nitrogen-acquisition ability of the species. Therefore, more research is needed to explain how some species are able to take more advantage of additional nutrients than others, and how this relates to their naturalization success.

Nitrogen (N) is a macronutrient essential for plant growth, and has been considered a crucial factor driving the increase in local abundance of naturalized alien plants (Seabloom et al. 2015; Liu et al. 2017a, b). Generally, species with high local abundances have large populations, which can produce and disperse higher numbers of seeds in the landscape, resulting in more populations and a larger geographical range (Primack and Miao 1992; Schupp et al. 2010; Ramírez-Rodríguez and Amich 2017; Liu et al. 2018). Indeed, a comprehensive assessment of the different dimensions of invasion success for the European alien seed plant flora showed that the maximum local abundance of alien plant species in vegetation plots is positively correlated with their naturalization extent (number of regions where naturalized; Fristoe et al. 2021). Consequently, N use and response is also frequently considered to drive global naturalization success of alien species (Dostál et al. 2013; Liu and van Kleunen 2019).

Nitrogen comes in different forms. Beside the the two inorganic N forms (ammonium [NH4+] and nitrate [NO3−]), it also comes in multiple organic forms. Actually, more than 20 forms of free amino acids in the soil are N sources for plants (Persson and Näsholm 2001). For some amino acids (e.g. glycine and histidine), it has been shown that their contribution to the total N take up could range from 0.5% for crops to 82% for arctic/alpine plants (Kielland 1994; Xu et al. 2006, 2008). Due to atmospheric N deposition and fertilizer run-off from agricultural land, as well as by changes in microbial activity induced by climate change, both the availability of soil N and the the relative amounts of the different N forms are becoming highly heterogeneously distributed (Krupa 2003; Liu et al. 2016a, b; Homyak et al. 2021). It is frequently assumed that variation in preferences for various soil N forms could allow plant species to decrease their niche overlap, and to promote coexistence (McKane et al. 2002; Ashton et al. 2010). Following this logic, a species that can successfully invade, and thus coexist with the resident species, might be more promiscuous in the use of different nitrogen forms than less-successful alien species. In other words, those alien plants that can maintain a high performance under varying N-availability levels and take advantage of multiple N forms should be able to establish at more locations and thus have a larger non-native geographic range. This would also be in line with the long-standing idea that generalists, with a high environmental tolerance, have higher invasion potential than specialists (Baker 1965; Richards et al. 2006).

The preferences for various N forms are species-specific (Ashton et al. 2010; Boudsocq et al. 2012; Liu et al. 2017a, b; Liu et al. 2020). For example, the invasive species Mikania micrantha, Ipomoea cairica, Wedelia trilobata and Bidens pilosa performed better when supplied with NO3− than with NH4+ (Chen and Chen 2019). However, the invasive species Flaveria bidentis is able to use NO3− and NH4+, depending on which form is most available in the environment, and this promiscuity probably has contributed to its dominance in many communities (Huangfu et al. 2019). To test whether a preference for a certain N form or promiscuity generally contributes to naturalization success at the global scale, we need studies that compare successful with less successful alien plant species. However, there are many different factors that might determine the naturalization success of a species. Variation in naturalization success caused by these other factors can be reduced by comparing species that are native to the same region but differ in their invasion success elsewhere (i.e. the source-area approach) (Pyšek et al. 2004; van Kleunen et al. 2010; Liu and van Kleunen 2019). Furthermore, as species that are common in their native range might have a higher chance to be picked up and introduced elsewhere (e.g. van Kleunen et al. 2007; Pyšek et al. 2009), one can reduce this source of variation by selecting species that are equally common and occur in similar habitats in their native range.

To test whether preferences for certain N forms or promiscuity with regard to N forms could drive variation in naturalization success of alien species, we exposed 22 common Central European herbaceous grassland species that differ in their global naturalization success to six different N treatments. The six N treatments included one low and five high N-availability treatments: (1) low availability with equal amounts of nitrate-nitrogen (NO3−-N), ammonium nitrogen (NH4+-N), glycine (Gly-N) and histidine (His-N). (2) high availability with equal amounts of NO3−-N, NH4+-N, Gly-N and His-N, (3) high availability of NO3−-N only, (4) high availability of NH4+-N only, (5) high availability of Gly-N only, (6) high availability of His-N only. We compared how biomass and root allocation responses to the N treatments related to the global naturalization success of the species. Specifically, we tested the following questions: (i) Do widely naturalized species overall show a stronger response to increasing N availability than less widely naturalized species? (ii) Do the preferences for the different inorganic and organic N forms differ between widely and less widely naturalized species? (iii) Are widely naturalized species overall more promiscuous with regard to the different N forms than less widely naturalized species?

Materials and methods

Study species

To increase our ability to generalize the results on N-preference differences among species varying in their global naturalization success (van Kleunen et al. 2014), we selected 22 herbaceous plant species from eight families, and with different life spans (Table S1). All 22 species are common natives in Germany (occuring in at least 1928 of the 3000 German grid cells; Table S1), but have become naturalized to different extents in the rest of the world. All the naturalized species exhibit similar Ellenberg N‐indicator values (Fig. S1; Table S1), reflecting the N‐conditions in the natural habitats of the species in Europe (Ellenberg 1974). This measure is pertinent as species originating from N‐rich habitats are likely to have a higher naturalization success (Dostál et al. 2013). We determined the number of regions in which each species has become naturalized from the Global Naturalised Alien Flora (GloNAF) database, version 1.2 (van Kleunen et al. 2019), which includes data on naturalization success of 13,939 plant species across a total of 1029 regions. The number of GloNAF regions in which the selected species are recorded as naturalized ranged from 1 to 283 (Table S1), thereby including some of the globally most widely naturalized species (Pyšek et al. 2017). The number of naturalized regions of the 22 species is also strongly positively correlated with the number of countries where the species are considered invasive according to the Global Register of Introduced and Invasive Species (Pagad et al. 2022; r = 0.77, p < 0.001; Fig. S2). This implies that the more widely naturalized a species is, the higher its invasiveness worldwide. The seeds of all but two study species, which were collected from a field site in Konstanz, were bought from a commercial seed company (Rieger-Hofmann GmbH, Germany; Table S1). The company produces native seeds, representative of natural genotypes, for grassland-restoration and agricultural purposes (i.e. sowing of pastures and meadows).

Pre-cultivation and experimental set-up

To test whether the global naturalization success of the study species is related to their N-preferences, we did a greenhouse experiment in the botanical garden of the University of Konstanz (Germany). On 18 and 19 May 2020, seeds were sown into trays (12.0 cm × 12.0 cm × 4.5 cm) filled with potting soil (Topferde®, Einheitserde Co., Sinntal‐Altengronau, Germany; pH 5.8; 2.0 g/L KCl; 340 mg/L N; 380 mg/L P2O6; 420 mg/L K2O; 200 mg/L S; 700 mg/L Mg) for each of the 22 species separatately. We then placed the trays in a greenhouse at a temperature between 22 and 28 °C, and a day:night cycle of 16:8 h.

On 2 June 2020, two weeks after sowing, we selected 18 similarly sized seedlings per species, and transplanted each of them separately into circular 2-L plastic pots (i.e. one individual per pot) filled with a 1:1 nutrient-poor mixture of sand and fine vermiculite. In total, the experiment included 396 pots (i.e., 18 pots per species × 22 species), which were randomly assigned to positions on four greenhouse benches. To test whether responses to the amount of N and the different forms of N vary among the study species and relate to their naturalization success, we assigned the 18 pots of each species to six different N treatments. In other words, there were three replicate pots per species under each N treatment. As we were interested in promiscuity to different N forms between widely naturalized and less widely naturalized species, rather than in differences among the individual species, we had maximized the number of species over the number of replicate pots per species (van Kleunen et al. 2014). Although in nature, plants frequently grow in competition with other plants and have their roots intertwined, we chose to grow each plant individiually so that we were able to harvest all belowground parts and thus could determine total biomass production and root allocation responses to the varying N conditions.

The N treatments started two weeks after transplanting, and lasted for eight weeks. The six N treatments included one low N-availability treatment and five high N-availability treatments. Each week, we applied the six different N treatments using six modified Hoagland solutions that only differed from each other in the amount or form of N (for details, see Method S1). In other words, all nutrient solutions contained the same amounts of the other nutrients (e.g. P, K). Since the study species have become naturalized in diverse habitats across various regions of the world, it is unfeasible to manipulate all different N forms they encounter in naturalized soil. Therefore, we focused our study on two common inorganic N forms (ammonium [NO3−-N], stock-solution pH = 5.26; nitrate [NH4+-N], pH = 5.05) and two common organic forms (glycine [Gly-N], pH = 5.35; histidine [His-N], pH = 7.30). The low N availability treatment (further referred to as ‘low mixed N’) included a 1:1:1:1 mixture of NO3−-N, NH4+-N, Gly-N and His-N. For the five high N-availability treatments, the N forms differed: (i) a 1:1:1:1 mixture of NO3−-N, NH4+-N, Gly-N and His-N (further referred to as 'high mixed N'), (ii) only NO3−-N, (iii) only NH4+-N, (iv) only Gly-N, (v) only His-N. Because the charge of amino acids might determine their diffusion rates in soil (e.g. Homyak et al. 2021), we chose glycine, a neutrally charged amino acid, and histidine, a positively charged amino acid. We supplied 40 ml of each of the nutrient solutions to each pot once a week, so that 0.24 mmol and 2.4 mmol of N were provided each time for low mixed N and the five high N availability treatments, respectively. In other words, for the high nitrogen treatments, the nitrogen fertilizer applied during the experiment was equivalent to a total of 118.5 kg N per ha. This amount is slightly higher than the annual atmospheric N deposition in highly industrialized countries and comparable to the levels of agricultural fertilizer applications in many parts of the world (Galloway et al. 2008).

During the entire experiment, we kept all pots at temperatures between 22 and 28 °C, and provided supplemental lighting to extend the daily light period to 14 h. We watered each pot when the soil looked dry, and supplied the same amounts of water to all plants each time. To avoid that the nutrients leaked from the pots after watering or nutrient supply, we put a plastic dish under each pot.

Measurements

To be able to account for variation in initial sizes of the plants in the analyses, we counted, at the start of the experiment, the number of true leaves, and measured the length and width of the longest leaf on each plant. Based on these measurements, we calculated a proxy of initial leaf area as the length × width of the largest leaf × the number of true leaves. On 5 August 2020 (i.e. 8 weeks after the start of the N treatments), we assessed survival of the plants, and then started to harvest the ones that had survived. As 35 plants died during the experiment (Table S1), we finally harvested a total of 361 pots instead of 396 pots. We first harvested the aboveground biomass of each plant, and then washed their roots clean of substrate. The entire belowground biomass harvest took three days, from 6 to 8 August 2020. All aboveground and belowground biomass was dried at 70 °C for 72 h and then weighed. Based on the final aboveground and belowground biomass, we calculated the total biomass and root weight ratio (belowground biomass/total biomass) for each plant.

Statistical analyses

To test how survival related to naturalization success (i.e., the number of regions in which the species naturalized) and the different N treatments, we fitted a binomial generalized linear mixed-effects model using the glmer function in the package "lme4" (Bates et al. 2015) in R 4.0.3 (R Core Team 2020). Similarly, to test how total biomass and root weight ratio were related to naturalization success and the different N treatments, we fitted linear mixed-effects models using the lme function in the package “nlme” (Pinheiro 2011).

Survival, total biomass and root mass fraction of plants were the response variables in the models. To meet the normality assumption, total biomass was cubic-transformed. We included naturalization success (i.e. the number of naturalized regions per species), the different N treatments (i.e. low mixed N, high mixed N, high NO3−-N, high NH4+-N, high Gly-N and high His-N) and their interactions as fixed effects in the models. To test more specifically which of the N treatments differed from each other, we created a priori five orthogonal contrasts by coding dummy variables (Schielzeth 2010, Table S2): the mean of low mixed N vs. the mean of all high N treatments (Nlow-high), the mean of high mixed N vs. the mean of the non-mixed high N treatments (Nmix-nonmix), the mean of the two high inorganic N treatments vs. the mean of the two high organic N treatments (Ninorganic-organic), the mean of high NO3−-N vs. the mean of high NH4+-N (NNO3-NH4), the mean of high Gly-N vs. the mean high His-N (Ngly-his). Because variation in initial plant size might contribute to differences in final plant performance, we also added initial leaf area as a scaled natural‐log‐transformed covariate in the models. To account for phylogenetic non-independence of species and for non-independence of replicates of the same species, we included species nested within family as random factors in all models.

As the homoscedasticity assumption of linear mixed models was violated, we included variance structures in the models to allow different variances per species using the function "varIdent" in the R package nlme (Pinheiro 2011). As species with different naturalization success might vary in their responses to the N treatments, we also ran models with random slopes for family and species with respect to N treatment. However, because this led to a singular fit for the survival model, and increased the AIC-values (i.e. decreased model fit) for the total biomass model and the RWR model, we did not include the random slopes in the final models. In all models, we considered an estimated parameter as significant when its 95% confidence interval did not overlap zero. In addition, we also extracted the χ2 and p values of each parameter using the Anova function with type-III errors from the R package car (Fox and Weisberg 2018).

To assess whether promiscuity to the different N forms was related to naturalization success of the species, we needed an index indicating whether the biomass responses of a species to the different N forms was relatively constant (indicating promiscuity) or very variable. We therefore first calculated for each species in each of the five high N treatments the response ratio of the average biomass in the respective high N treatment vs. the average biomass of the species in the low N treatment. Then as measure of how variable this response was, we calculated the standard deviation (SD) across the five response ratios of each species. As the SD of the response ratios only considers the variation in the response ratios and not the magnitude, we additionally calculated the coefficient of variation (CV) as the SD of the response ratio divided by the mean value of the response ratio. A low CV value would indicate promiscuity as low values arise due to low variation (small SD) and/or a high mean of the response ratio (i.e. a strong response to N availability). Finally, to test whether the SDs and C vs. of the response ratios were related to naturalization success of the species, we calculated Pearson’s correlation coefficients. As the choice of p-value cutoffs for significance testing is arbitrary, we followed the suggestion of Muff et al. (2022) to translate the p-values into the language of evidence.

Results

Of the initial 396 plants, 35 (8.8%) had died by the end of the experiment. Survival was not related to naturalization success of the species (Table S3). However, we found moderate evidence that survival was lower in the high N treatments than in the low N treatment, and was lowest when plants received N in organic form as glycine (Table S3, Fig. S3).

Among the surviving plants, and averaged across the six N treatments, there was no evidence that total biomass was related to naturalization success of the species (Table 1; Fig. S4A). Averaged across all species, plants produced more biomass (+ 159.7%) when grown under high N levels than under low N levels (Fig. 1A). The response patterns to nitrogen addition were similar for legume and non-legume species (Fig. S5). The increase in biomass production in response to low vs. high N was similar for the widely and less widely naturalized species (Fig. 1A). In other words, the 95% CIs of the Nat × Nlow-high coefficient estimate overlapped with zero (Table 1). Among the high non-mixed N treatments, plants also produced more total biomass (+ 73.4%) when N was provided in inorganic form than when provided in organic form (Table 1; Fig. 1A, B). There was moderate evidence that the increase in biomass production in response to inorganic vs. organic N was larger for the widely naturalized species than for the less widely naturalized species (see the Nat × Ninorganic-organic interaction in Table 1; Fig. 1B). Furthermore, there was moderate evidence that widely naturalized species preferred high glycine conditions over high histidine conditions, and that the less widely naturalized species has no preference for any of the two organic N treatments (see the Nat × Ngly-his interaction in Table 1; Fig. 1C).

Table 1 Results of linear mixed-effects models testing the effects of naturalization success (number of regions where the species is naturalized), nitrogen treatment and their interaction on total biomass (cubic-root-transformed) and root weight ratio
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Fig. 1
figure 1

Total biomass (cubic transformed) of the 22 study species in response to the different N treatments. A shows boxplots for the one low and five high N treatments. The boxes indicate the interquartile range around the median (thick horizontal line), whiskers extend to 1.5 times the interquartile range, and open circles indicate outliers. B illustrates how the difference in growth on inorganic (NO3−, NH4+) vs. organic (glycine, histidine) high N changes with naturalization success of the species, and C illustrates how the difference in growth on glycine vs. histidine changes with naturalization success of the species. In panel A, the hierarchical contrasts between treatments are indicated with horizontal lines. The significance of the main effect of the contrast is displayed next to the respective contrast line before the slash, while the significance of its interaction with naturalization success is indicated after the slash. A plot that shows plant biomass vs. naturalization success for each of the N treatments is provided in Fig. S4A

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The root weight ratio of plants was also not related to naturalization success of the species (Fig. S4B), and was decreased (− 33.1%) when plants were grown at high N levels (Fig. 2A). Averaged across all species, there was no evidence that root weight ratio varied among the different high N treatments (Table 1; Fig. 2A, S3B). There was moderate evidence that while root weight ratio decreased with increasing naturalization success for the high N mixture treatment and the high glycine treatment, it increased for the other high N treatments (Table 1, Fig. 2B–E, Fig. S4B). Moreover, there was moderate evidence that the root weight ratio increased more strongly with increasing naturalization success for the inorganic than for the organic high N treatments.

Fig. 2
figure 2

Root weight ratio (RWR) of the 22 study species in response to the different N treatments. A shows boxplots for the one low and five high N treatments. The boxes indicate the interquartile range around the median (thick horizontal line), whiskers extend to 1.5 times the interquartile range, and open circles indicate outliers. B illustrates how the difference in RWR between the low vs. high N mixture treatments changes with naturalization success of the species, C illustrates how the difference in RWR between the high N mixture vs. high N nonmixtures (NO3−, NH4+, glycine, histidine) treatments changes with naturalization success of the species, D illustraste how the difference in RWR between inorganic (NO3−, NH4+) and organic (glycine, histidine) high N treatments changes with naturalization success of the species, and E illustrates how the difference in RWR between the glycine and histidine high N treatments changes with naturalization success of the species. In panel A, the hierarchical contrasts between treatments are denoted by horizontal lines. The significance of the main effect of the contrast is displayed next to the respective contrast line before the slash, while the significance of its interaction with naturalization success is indicated after the slash. A plot that shows RWR vs. naturalization success for each of the N treatments is provided in Fig. S4B

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The species’ standard deviation and coefficient of variation for the response ratios of biomass in the high N treatments vs. biomass in the low N treatment were not significantly related to the naturalization success of the species (SD: r = 0.219, n = 22, p = 0.328; CV: r = 0.139, n = 22, p = 0.526; Fig. 2). In other words, there was no evidence that promiscuity to the different N forms was related to naturalization success (Fig. 3).

Fig. 3
figure 3

Scatterplot of the standard deviation (SD; A) and coefficient of variation (CV; B) of the response ratios (RR) of the five different high N treatments relative to the low N treatment of each species vs. the naturalization success of the species. A low SD and a low CV would indicate that a species responds similarly to the different N forms (i.e. that it is promiscuous). The correlations of naturalization with SD (r = 0.09, n = 22, p = 0.690) and CV (r = 0.139, n = 22, p = 0.526) of the response ratios were not significant

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Discussion

We tested whether preferences for and promiscuity with regard to different N forms of common Central European plant species were related to their naturalization success elsewhere. Not surprisingly, plants increased their performance when they had access to more N. However, we found that they benefited more from additional inorganic N than from organic N, and that high levels of glycine even increased mortality. The widely naturalized species did, averaged over the different N forms, not show a stronger response to increasing N availability than less widely naturalized species. Moreover, neither average biomass production nor promiscuity to different N forms was related to naturalization success of the species. However, the biomass response to inorganic N was, relative to the biomass response to organic N, and particularly to glycine, stronger for the widely naturalized species than for the less widely naturalized ones. These results indicate that naturalization success is not driven by N promiscuity, but might be partly driven by a species’ ability to take advantage of increased inorganic N.

Overall, plants increased their biomass production when growing under high N conditions relative to when grown under low N. This is not surprising because N availability is generally a limiting factor for plant growth (LeBauer and Treseder 2008). We also found that plants allocated less biomass to their root systems under high N conditions than under low N. This is in line with the predictions of resource-limitation theory, which states that plants should invest relatively more biomass into those structures that allow them to acquire more of the most limiting resource (Bloom et al. 1985; Poorter et al. 2012; Liu et al. 2016a, b; Liu and van Kleunen 2019). To assess whether the species can actually use each of the different N forms, we focused in this study on the single N forms and a mixture with equal ratios of the different N forms at both low and high levels. However, as in nature the different N-forms may occur at varying ratios, future studies should assess plant responses to a broader range of combinations and ratios of different N forms.

Numerous studies have shown that species that are more successful, either as natives or aliens, take more advantage of additional nutrients (Dawson et al. 2012b; Parepa et al. 2013; Liu and van Kleunen 2017). We found moderate evidence that widely naturalized species, relative to less widely naturalized species, decreased their biomass allocation to the root system more strongly in response to high N when compared to the low N-mixture treatment. However, this difference in root-allocation response among species varying in global naturalization success was apparently not large enough to result in a difference in overall biomass response to high N availability. The lack of a relationship between the naturalization success and the biomass response to high N availability could partly reflect that we only used species that are relatively successful in their native range and that some of them have not reached their full global naturalization potential yet. It could also be that the native genotypes that we used in our experiment are not representative for the genotypes that have become naturalized. Regardless of the exact reasons, futher studies should consider commonness in the native range and include a broader genotypic diversity of the study species. The lack of an association between global naturalization success and the response to high N availability, however, corroborates one of our previous studies, which showed that global naturalization success of Central European plants is not related to their N-acquisition ability (Liu and van Kleunen 2019). However, although we did not find a clear relationship between naturalization success and the overall response to an increased N availability, we found that biomass of the widely naturalized species increased more strongly in response to the inorganic N forms than was the case for the less widely naturalized species. So, the response to N addition might matter for naturalization success, but only if one considers inorganic N, which arguably are the most important N forms for plant growth.

While it was long thought that plants take up N exclusively in inorganic form, evidence is accumulating that many species are able to take up organic N forms (Kielland 1994; Persson and Näsholm 2001; Näsholm et al. 2009; Ashton et al. 2010; Boudsocq et al. 2012; Liu et al. 2017a, b; Homyak et al. 2021). Indeed, we found that most species increased their biomass when they were provided with the amino acids glycine or histidine instead of a low-N mixture. Nevertheless, plants benefited more from inorganic nitrogen (NO3−, NH4+) than from organic N. The latter is in line with previous studies, and might indicate that inorganic-N is the dominant N form in nature to which most species have adapted (Liu et al. 2017a, b).

Although most plant species produced more biomass in the high organic N treatments than on the low-N mixture, this does not necessarily mean that the plants took up the amino acids. Microbial turn-over of free amino acids is relatively rapid in natural soils. For example, it has been estimated that the half-lives of glycine and leucine in the field are normally less than 24 h (Henry and Jefferies 2003). Given the relative sterility of the sand:vermiculite mixture that we used in the present study, we expect that microbial conversion of organic to inorganic N was relatively limited. Still, we cannot fully exclude the possibility that some of the organic N in our treatments was mineralized by microbes, and that the plants took up the resulting inorganic N. However, if that would have been the case, we would expect that all species would have benefited from organic N addition and subsequent mineralization. Because there were also several species that produced less biomass in response to organic N additon (Fig. 1A and B), and because survival was lowest in the high glycine treatment (Fig. S3), it seems likely that the species that benefited took up the organic N forms directly.

Soil pH can also have a strong effect on plant growth. As the different N forms may inherently result in different soil pHs, the direct effects of the different N forms and the indirect effects through changes in soil pH cannot be separated in our study. So, it could be that the lower survival and biomass production of plants in the high glycine treatment than in the high histidine treatment, could be due to the lower pH values of glycine (stock-solution pH = 5.35) than histidine (pH = 7.30). However, this seems unlikely because the stock-solution pH of glycine was similar to the ones of nitrate (pH = 5.05) and ammonium (pH = 5.26), which were the most beneficial for plant performance. A case study on Ambrosia artemisiifolia indicated that plants grown in various natural soils with pH values between 5 and 7 had similar germination rates and growth performance (Gentili et al. 2018). Therefore, the different effects of glycine and histidine are unlikely caused by pH differences, although we cannot exclude that the differences in responses to glycine and histidine of the widely and less widely naturalized species (Fig. 1C) are caused by differences in pH sensitivities of the study species.

As mentioned above, the widely naturalized species benefited more from inorganic N, relative to organic N, than less widely naturalized species. In particular, the widely naturalized plants benefited less from the addition of glycine, relative to histidine. These biomass-response patterns are consistnet with root-weight-ratio responses to these different N treatments. This is not suprising because the more biomass is allocated to the root system, the more nutrients can be taken up and the more a plant can grow. Overall, these results suggest that there is a trade off between species’ responses to inorganic and organic N forms, and that those that benefit the most from inorganic N have the highest naturalization potential. Additionally, previous studies have shown that the different inorganic-N forms, NO3− and NH4+, could affect plant performance differently (Ashton et al. 2010; Huangfu et al. 2016; Chen and Chen 2019; Liu et al. 2020). We found that performance of our study species overall did not significantly differ between the NO3− and NH4+ treatments. Although plant responses to different N forms in nature may also depend on competition (Ashton et al. 2010), our findings highlight that changes in the NO3−/NH4+ ratio induced by atmospheric N deposition (Boxman et al. 2008) might not affect the absolute performance of the plants.

In all five high N treatments, plants invested on average less biomass in their roots than in the low N treatment. Averaged across species, root weight ratios did not differ among the different high N treatments. However, we found that in the high N mixture and high glycine treatments, the relative biomass investment into roots declined with the extent of naturalization of the species, while the reverse was true for the other N treatments. The reasons for these differences are unknown. However, the fact that the widely naturalized species produced low root weight ratios in the high glycine treatment might have contributed to their low biomass production in this treatment. On the other hand, the widely naturalized species invested more biomass in roots, and also produced more biomass than the less widely naturalized species in the high histidine treatment. This shows that plants do not benefit equally from all organic N forms, and this might be related to the charges of the amino acids and the differences in pH (Homyak et al. 2021). Although glycine is often considered to be a common amino acid in soils, the same can be true for histidine, as well as for amino acids that we did not included in our experiment (e.g. lysine, arginine and serine) (Lipson and Näsholm 2001; Tian et al. 2020). Future studies on N responses of plant species differing in their naturalization success should also consider these other amino acids as well as other organic N forms that could be taken up by some plants, such as urea and quaternary ammonium compounds (Warren 2013).

Conclusions

Our study is, to the best of our knowledge, the first multi-species experiment, using the source-area approach, that compared preferences for specific N forms and promiscuity to different N forms among species differing in their global naturalization success. While the widely naturalized alien species were not more promiscuous than the less widely naturalized species, they took more advantage of the inorganic-N forms, nitrate and ammonium. As these forms have globally increased due to atmospheric nitrogen deposition, our results suggest that one does not have to be a N generalist in order to become widely naturalized around the world. Instead, it helps to specialize in the most common forms of N available to plants.