Identifying limitations for invasion: the effect of phosphorus availability on the growth of the non-native tree, Tipuana tipu

Study species

Tipuana tipu (Benth.) Kuntze (Fabaceae) is a medium–large tree, with deciduous bi-pinnate leaves, yellow flowers and one to five seeds held within a woody samara, or winged achene (Martins and Oliveira 2001; Cruz et al. 2002). Common names include Bolivian rosewood, pride of Bolivia, tipa or racehorse tree (the latter on account of its rapid growth rate). The climate in the native range varies from semi-arid to subtropical (Antezana and Navarro 2002; Brown et al. 2007). In its native range, it is a common or dominant component of several vegetation types (Navarro and Ferreira 2004).

There are multiple records of naturalisation, predominantly in countries with a Mediterranean climate (Trudgen 2020). Young seedlings have been observed at numerous locations near cultivated trees in south-western Australia; however, the species is not known to have naturalised outside urban environments in this region (Trudgen 2020). The invasion-pathway framework of Blackburn et al. (2011) was used to define invasive, and the framework of Webber and Scott (2012) to define non-native for this study.

Growing conditions

A stratified randomised-block design pot experiment was established in a glasshouse, with the intention of simulating growing conditions in south-western Australia. The experiment was conducted from October 2012 to March 2013 in a glasshouse at The University of Western Australia (31°59′S, 115°49′E), and the plants were harvested 15 weeks after sowing. In the glasshouse, the mean monthly minimum temperature was between 14.3°C and 19.7°C, and the mean monthly maximum temperature was between 29.1°C and 32.0°C. Coarse quartz river sand, sieved to 2 mm, was used as the growth medium because (a) naturally occurring soils in south-western Australia tend to be dominated by sand (Lambers et al. 2010), and (b) the low nutrient concentrations applied in this study are not possible to control in soils with high organic matter. The soil was washed with tap water and oven dried prior to use, and had <2 μg P g⁻¹ dry soil prior to nutrients being added to the soil. Nutrients (Table 1; consistent with Pearse et al. 2006; Pang et al. 2010; Suriyagoda et al. 2010) were mixed by a cement mixer prior to about 3 kg of the mixed sand being added to each of 72 pots. Pots were 125 mm × 210 mm white PVC pipe, with 1 mm flyscreen mesh taped across the bottom. To assist aeration, the pots had a v-shaped plastic frame positioned between the pot and the drip tray. Phosphorus was added to the sand in each pot (as dissolved KH₂PO₄, with KCl at an appropriate rate to balance K across the treatments) at one of the eight treatment levels of 0, 5, 10, 40, 80, 160, 320, 640 μg P g⁻¹ dry soil and the pots were watered every 1–3 days to maintain 70% field capacity. The dried fruit (samaras) were X-rayed (Faxitron MX-20 Specimen Radiography System, Tucson, AZ, USA) prior to sowing, to determine the number of viable seeds per samara, with viability assessed visually on the basis of the size, shape and fill of the seed(s) within the samara. The samaras used for the experiment were randomly chosen from among the samaras with two viable seeds, and the wings of the samara were removed with scissors prior to planting. Two samaras (and therefore a total of four seeds, visually similar by X-ray) were sown per pot. Germination was recorded daily, and plants were thinned to one plant per pot 3 weeks after sowing (as per Poorter and Garnier 1996; Poorter et al. 2012).

Plant measurements

Non-destructive measurements (stem height and leaf number, and observing if the leaf at each leaf node was immature, mature or senesced) were recorded on the day of seedling thinning, and remeasured three times (at 4-weekly intervals) thereafter, including on the day of the final destructive harvest. Senesced leaves (if any) from each plant were collected and oven dried immediately, with all senesced leaves from each plant combined at the end of the experiment to allow resorption of nutrients to be measured. In the final destructive harvest, each plant was severed at ground level. Leaves were removed from the stem and roots were gently washed free of the sand by using a light stream of water and a 2 mm sieve plate to prevent fine roots from being washed away. Nodules were counted, removed from the roots and stored for oven drying. The stem was dried immediately, and the leaves stored at 2°C until they were scanned. Fresh leaves were scanned (RGB TIFF; 300 dpi) on an Epson 750 flatbed scanner and leaf area was calculated using the LEAF GUI (Price 2012). All plant material was dried at 68°C for a minimum of 7 days to obtain dry weight.

Chemical analyses

Plant materials (leaves, stems, roots, senesced leaves and thinned germinants) were individually ground using an electric coffee grinder before subsampling for chemical analysis. Nodules were excluded from analyses because of a lack of material across treatment groups. Thinned germinants were used as a proxy for seed reserves, because it was not possible to safely extract individual seeds from the woody samaras. Phosphorus was analysed (with a precision of 0.01 mg g⁻¹) following the protocol for small samples in Fourqurean et al. (1992). Nitrogen was analysed using the Leco Total Nitrogen method (with a precision of 0.1% by weight) for the Leco Combustion analyser (TruSpec CN Carbon Nitrogen Determinator Instruction Manual Software, ver. 1.6 x., Part No. 200-288, March 2006). Phosphorus-resorption efficiency (PRE) and nitrogen-resorption efficiency (NRE) were calculated as the change in nutrient concentration between mature green and fully senesced leaves (nutrient concentration in green leaves minus the nutrient concentration in senesced leaves, all divided by the nutrient concentration in green leaves and multiplied by 100, as per Killingbeck 1996). Values for N and P in the fully senesced leaves are equivalent to values of nutrient-resorption proficiency (as per Killingbeck 1996).

Data analysis

Non-destructive measurements were analysed using repeated-measures ANOVA and Tukey’s HSD test (P < 0.05). Harvest results and chemical analysis results were analysed using two-way ANOVA and Tukey’s HSD test (P < 0.05). Five plants had atypical growth patterns (branching owing to thrip damage, or unusually early flower production) and were excluded from analyses. All analyses were undertaken in R ver. 3.5.1 (R Core Team 2018) and the broom, Cairo, car, emmeans, facetscales, lattice, modelr, multcompView, nlme, rcompanion and tidyverse packages (Sarkar 2008; Graves et al. 2015; Wickham 2017, 2019; Fox and Wesiberg 2019; Lenth 2019; Mangiafico 2019; Oller Moreno 2019; Pinheiro et al. 2019; Robinson and Hayes 2019; Urbanek and Horner 2019).

Plant response to soil P availability

Seed P reserves are an important resource for seedling establishment, and are considered a strategy of increasing seedling survival in nutrient-poor soils (Kuo et al. 1982; Denton et al. 2007). Growth of T. tipu seedlings up to 7 weeks after sowing appeared to be driven by nutrient reserves from the seed endosperm, because plant height and the number of mature leaves per plant were consistent across treatments up to this time. Germinant P concentration of T. tipu (measured in lieu of seeds) was greater than the average concentration recorded for seeds from species adapted to the nutrient-poor soils of south-western Australia (13.2 ± 0.8; Kuo et al. 1982; Groom and Lamont 2010; Hayes et al. 2021); however, the total P content of T. tipu seeds was much lower than recorded for those seeds, owing to lower total germinant weight. This indicates that T. tipu is not adapted to relying on its seed P reserves to support a long period of establishment or survival as a seedling during periods of enduring resource limitation.

Soil nutrient availability has a strong influence on plant growth, and the T. tipu seedlings in our study showed a strong response to soil P concentration. Plants grown at very low soil P concentrations (≤5 μg P g⁻¹ dry soil) began to show signs of P deficiency, leaf senescence, very slow growth and showed declining vigour across all replicates after 7 weeks. When these plants were harvested, it became clear that they had invested heavily in root growth, which is commonly observed in plants grown in conditions of P deficiency (e.g. Wang et al. 2015) and corresponds with a growth strategy to locate soil nutrient reserves and increase root surface area for absorption of P (Vance et al. 2003; Lambers et al. 2006). Therefore, we conclude that plants grown at ≤5 μg P g⁻¹ dry soil had insufficient P available to sustain vigorous growth beyond approximately 7 weeks.

Plants grown at slightly higher soil P concentrations (10 μg P g⁻¹ dry soil) were able to sustain growth for longer; however, they still appeared to exhaust their limited P supply and had signs of P deficiency after 11 weeks. Although the P concentration in senesced leaves (a measure of nutrient-resorption proficiency) of plants grown at ≤10 μg P g⁻¹ dry soil was somewhat higher that that of plants native to the nutrient-poor soils of south-western Australia (Hayes et al. 2021), the P-remobilisation efficiency (34–67%, PRE) was similar to the lower end of P-remobilisation efficiency for those plant species (27–82%, Denton et al. 2007; approximately 20–80%, He et al. 2011). Although it is possible that some leaching occurred from the senesced leaves prior to collection, they were collected as soon as possible to minimise this possibility. Although the plants in our study grown at ≤10 μg P g⁻¹ dry soil resorbed significant P from the leaves prior to leaf abscission, they still had significantly less P in their plant organs than did healthy T. tipu plants grown at higher soil P concentrations. Although P deficiency is uncommon in young P-rich soils, plants in our study were well supplied with N and other baseline nutrients, and P was the limiting nutrient in the river sand used in our experiment (Lambers et al. 2010).

The optimal soil P concentration for growth of T. tipu plants in our study was between 40 and 80 μg P g⁻¹ dry soil, with plants in these treatments having the greatest dry mass, number of mature leaves and plant height. This optimal soil P concentration is much higher than that for many species adapted to soils with low P concentrations (e.g. Williams et al. 2019), and is most likely to reflect the soil conditions in the native range of southern Bolivia and northern Argentina (Brown et al. 2007; Atahuachi et al. 2012). Plants from one of the treatment groups with optimal soil P concentration (80 μg P g⁻¹ dry soil) had a significantly higher proportion of nodules (g g⁻¹ total biomass) than did those from the other treatment groups, and had significantly greater biomass and height than did plants from other treatments. This finding indicated that these plants were N limited rather than P limited and invested in nodule growth to access additional N supplies (Sprent and Thomas 1984). Plants in this treatment resorbed P and N from the leaves prior to senescence, and it appears likely that the greatly increased nodulation on these plants increased the plants overall demand for P, because increased N availability enhances the demand for other nutrients (Eller and Oliveira 2017; Valliere et al. 2017).

Across the treatments, foliar, stem and root P concentrations increased and PRE decreased as soil P concentration was increased, being consistent with previous studies (e.g. He et al. 2011). Foliar P concentrations were relatively high for T. tipu when compared with those for plants adapted to the nutrient-poor soils of south-western Australia (0.14–0.27 mg g⁻¹, Denton et al. (2007); <1 mg g⁻¹, Hayes et al. 2021); however, they were low in comparison with global foliar P concentrations for deciduous angiosperms (0.75–10.9 mg g⁻¹ compared with 15.5 mg g⁻¹ globally; Vergutz et al. 2012).

Plant growth of T. tipu in our study was negatively affected by surplus P availability at high soil P concentrations (≥160 μg P g⁻¹ dry soil). Above the optimal soil P concentration range (between 40 and 80 μg P g⁻¹ dry soil), dry mass and leaf area decreased for each increase in soil P concentration, and plants at higher soil P concentration decreased their investment in belowground growth. This growth pattern was opposed to the pattern exhibited by plants when they are P limited (Lambers et al. 2006), and suggests that the plants obtained the nutrients required for growth from a small volume of soil. We found no evidence of the plants in our study down-regulating P-uptake capacity at the higher soil P concentrations, and the P concentrations in senesced leaves were within the range of P concentrations at which P toxicity typically develops (Aiello and Graves 1996; Shane et al. 2004; Lambers 2022). The negative PRE values for plants grown at very high soil P concentrations (≥160 μg P g⁻¹ dry soil) demonstrated that the plants were depositing P in these leaves prior to leaf senescence. Taken together, these findings provided strong evidence that the relative decrease in dry mass and leaf area of plants grown at high soil P concentrations was a result of P toxicity, rather than nutrient deficiency.

Nutrient use and invasive-species establishment

The nutritional requirements of non-native invasive species are an aspect of invasion ecology that has not been studied enough to draw out general patterns on the associated traits that lead to invasion success (Pyšek and Richardson 2007), despite the recognised importance of adequate nutrition for plant growth and survival. The establishment of seedlings is generally considered the most vulnerable developmental stage for a plant (Wilson and Witkowski 1998). Our study has demonstrated that the seedling growth of T. tipu is affected by soil P availability, and it is likely that soil P availability constrains the capacity of this introduced species to establish self-sustaining populations in regions with very low soil P concentrations (≤5 μg P g⁻¹ dry soil). These findings add to an increasing body of evidence (e.g. Witkowski 1994; Riley and Banks 1996; Burns 2004; Eskelinen and Harrison 2014; Hu et al. 2019) that suggests that a plant’s response to nutrients should be considered when assessing the invasion risk posed by a species, and when developing suitable management plans.

In our results, T. tipu plants grown at very low soil P concentrations (≤5 μg P g⁻¹ dry soil) were unable to sustain growth beyond 7 weeks, indicating that T. tipu will be very unlikely to survive to reach maturity, and even less likely to establish and form self-sustaining populations in regions with very low soil P concentrations, such as south-western Australia (Lambers et al. 2010). T. tipu plants grown at these very low soil P concentrations were small at the end of our study, with extremely low biomass in comparison with tree species native to the nutrient-poor soils of south-western Australia (Zhou et al. 2022). Increased height is associated with invasiveness (Pyšek and Richardson 2007) and the small stature of these plants is likely to make them vulnerable in the wild to mortality by a range of causes such as trampling, browsing, mowing, or targeted weed removal. Although we have not directly assessed competitive ability in this study, the low total biomass, short stature, and signs of P deficiency of T. tipu grown at very low soil P concentrations, in comparison with species adapted to nutrient-poor landscapes (e.g. Denton et al. 2007; He et al. 2011; Zhou et al. 2022), suggests that T. tipu has a low competitive ability against species that are adapted to low soil P concentrations. At optimal soil P concentrations (40–160 μg P g⁻¹ dry soil), T. tipu was capable of rapid growth across a range of measures (height, biomass, number of mature leaves), and the species’ competitive ability is likely to be greater at these soil P concentrations. This contrasts with seedlings from species that are adapted to nutrient-poor soils, which often have greater biomass at very low soil P concentrations and lower biomass when grown in higher soil P concentrations (Standish et al. 2007; Williams et al. 2019). Although plants in natural conditions would be subject to a range of other factors affecting growth, we expect that, in suitable climatic conditions, soils with P concentrations in this range (40–160 μg P g⁻¹ dry soil) may allow naturalised populations of T. tipu to establish. At quite high soil P concentration (640 μg P g⁻¹ dry soil), plants in our study showed signs of P toxicity, which would likely prevent the establishment of naturalised populations where high soil P concentrations occur. This concentration of soil P is extremely high for nutrient-poor landscapes such as south-western Australia, although it is possible that localised nutrient pollution may occur, with riparian areas at a particular risk of accumulating excess P; however, this soil P concentration may occur in soils in other regions around the world (Stock and Verboom 2012). Areas with unnaturally high soil P concentrations as a result of nutrient enrichment are of particular interest with regard to plant invasions, because the growth of native species may be suppressed, whereas non-native species may respond to the higher soil nutrient concentrations with increased growth and, therefore, have a competitive edge in such situations (Williams et al. 2019).

The consideration of nutrient requirements in invasion management is hampered by two main limitations, namely, the lack of data on nutrient requirements for individual species, and the lack of suitably scaled and up to date soil nutrient maps (see Michael et al. 2012; Matos-Moreira et al. 2017). Even though we have been able to describe the growth response of T. tipu seedlings across a broad range of soil P availabilities, our findings remain limited by the lack of suitable soil maps (Viscarra Rossel and Bui 2016; Kooyman et al. 2017), and we have been unable to map regions where very low soil P concentrations may prevent the establishment of non-native T. tipu populations (see Michael et al. 2012). Moreover, nutrient pollution of soils can circumvent this limit to invasion risk and is perhaps most likely in urban and agricultural environments, which have a high proportion of non-native species with the potential to become invasive (Kowarik 2011). The issue of scale and currency of soil maps will, therefore, be particularly important in these environments. Until these two limitations are overcome, it will be difficult to accurately account for nutrient requirements in species risk assessments and management plans at a large scale (Viscarra Rossel and Bui 2016). Nonetheless, soil nutrients remain a critical microhabitat consideration to assess the risk of non-native plants moving down the invasion pathway, particularly for microhabitats around cities where riparian areas and nutrient pollution are more frequent in the landscape.

Conclusions

We found that the growth of young T. tipu seedlings was significantly affected by soil P concentrations, with implications for their potential capacity to become invasive, particularly in landscapes with very low soil P concentrations (≤5 μg P g⁻¹ dry soil). The growth of young T. tipu seedlings was very slow at very low soil P concentrations, and the optimal soil P concentration range for growth was higher than that for plants adapted to low soil concentrations (e.g. He et al. 2011; Williams et al. 2019). The extremely poor vigour and growth of T. tipu plants at very low soil P concentrations, the low number of mature leaves on these plants, and the importance of height and rapid growth rates as traits of successful plant invasions, lead us to conclude that T. tipu will likely be constrained in establishing self-sustaining populations in locations with very low soil P concentrations. T. tipu plants exhibited signs of P toxicity beyond the optimal concentration of soil P, which may also constrain the invasive potential in areas with quite high soil P concentrations (≥640 μg P g⁻¹ dry soil). Our study contributes to the growing body of evidence that soil nutrient availability and soil nutrient requirements are an important aspect to consider when contextualising plant invasions. Such insight represents an important inclusion into invasive species risk assessments and management plans, particularly at a fine scale where microhabitats are a significant aspect of the landscape.