1 INTRODUCTION

An invasive species is an organism that is introduced, often with human assistance, to a region it did not previously inhabit, where it establishes a population that spreads independently (Simberloff, 2010). Arundo donax L., commonly known as giant reed, is a perennial invasive C3 grass that has been introduced to the southern part of the continental United States as well as Hawaii, Puerto Rico, and the Virgin Islands (USDA, 2022). Arundo donax is hypothesised to have originated in Asia and diffused into the Mediterranean, from whence it was introduced to North America in the early 1800s (Ahmad et al., 2008; Hardion et al., 2014). Genetic analysis of individuals in North America revealed that all 185 sampled A. donax, from 15 states and four populations in southern France, were genetically identical, except for one sample in Texas that exhibited a single mutation (Ahmad et al., 2008). Further investigation into the reproductive mechanisms of A. donax showed that the North American invader is sterile and incapable of reproducing via seed (Mariani et al., 2010). The spread of A. donax through North America can be attributed to human-mediated introductions and asexual vegetative reproduction via layering, rhizomes and fragmentation (Ahmad et al., 2008).

The fast growth of A. donax, along with its lack of significant natural enemies, has made the species an aggressive invader of riparian habitats in its introduced range. Arundo donax can grow up to 10 m tall, forming dense monotypic stands associated with lower species richness in stream banks and floodplains and has been linked to decreased streamflow in the Nueces River (Cushman & Gaffney, 2010; Jain et al., 2015). The species is typically found along disturbed stream beds, lakes and other wet areas and can grow 30–70 cm in height per week under ideal conditions (Bell, 1997; Perdue, 1958). The C:N ratio of A. donax tissues in California was found to be less than the range of C:N ratios considered favourable by aquatic herbivores (Mattson Jr, 1980; Spencer et al., 2005), which might make it a less desirable food source.

Arundo donax can be found in a wide range of soils, from dense clays to loose sand, and is tolerant of high salinity conditions (Di Mola et al., 2018; Perdue, 1958). Though capable of withstanding extreme drought and excessive soil moisture, A. donax seems to be most successful in areas with well-drained soil and ample moisture (Perdue, 1958). The performance related traits of A. donax, such as height and shoot survival, in Southern California have been found to be closely correlated to soil moisture, and in some cases disturbance (Quinn et al., 2007).

Though able to subsist in infertile conditions, past studies suggest A. donax performs best in high nitrogen conditions (Perdue, 1958). The plant seems to favour nitrogen delivered as NH₄⁺ or NH₄⁺ NO₃⁻ instead of solely NO₃⁻ (Tho et al., 2017). As is the case with many invasive species, A. donax shows higher biomass production and spread in response to increased nitrogen (Nackley et al., 2017; Quinn et al., 2007; Tho et al., 2017). Studies indicate that fast-growing invasive species like A. donax may gain a competitive advantage over native species when N is increased. This can lead to conditions in which invasive species are able to, through faster growth and larger size, outcompete native species for resources, mainly light (Mangla et al., 2011). This concept is supported by the success of many large-statured wetland species grown in high nitrogen conditions, such as Phragmites australis, Typha domingensis and Phalaris arundinacea (Martina & von Ende, 2012; Minchinton & Bertness, 2003). Studies of biomass allocation patterns in A. donax have yielded mixed results. Quinn et al. (2007) found that A. donax grown in pots treated with nitrogen allocated more biomass to aboveground biomass (AGB) and root tissues while Nackley et al. (2017) found that A. donax grown in CO₂ and N-enriched environments allocated more biomass to rhizomes. Studies to date have yet to address how different soil moisture regimes interact with nutrient loading to impact the competitive ability and resource allocation of A. donax.

The success and impact of invasive species is closely related to their biology as well as the attributes of the invaded ecosystem (Lonsdale, 1999). In a meta-analysis of traits related to invasiveness, van Kleunen et al. (2010) found that invasive plant species generally have higher values of performance-related characteristics, including but not limited to growth rate, size, and fitness. Furthermore, a meta-analysis of the traits of invasive plant species and their impact on the invaded ecosystem suggested that ecosystem productivity was dependent on height. Invasive species taller than 1.2 m significantly affected the productivity of plant and animal communities (Pyšek et al., 2012). However, these results are strongly context dependent and may not necessarily be true for all plants and ecosystems. Biomass allocation patterns may also play an essential role in establishment success and differ between native and invasive species. A study comparing two invasive and two native grass species found that the invaders tended to allocate more biomass to roots while natives allocated more biomass to the crown (Dong et al., 2014). Phalaris arundinacea was found to allocate more biomass to aboveground organs in high light, high soil moisture and high nitrogen conditions, however in high light and low nitrogen conditions increased soil moisture was associated with lower AGB (Martina & von Ende, 2012).

There is conflicting evidence surrounding differences in physiological attributes, such as water use efficiency (WUE) and photosynthetic rate, between invasive and native species. WUE has been found, under resource-limited conditions, to be higher in native species than in their invasive counterparts (Matzek, 2011). However, many studies have shown little difference between the physiological attributes of invasive and native plant species. A comparison of invasive and native genotypes of Phalaris arundinacea revealed that physiological characteristics of native and invasive genotypes were not significantly different, except for photosynthetic rate and stomatal conductance being higher in the native genotype (Brodersen et al., 2008). Analysis of A. donax litter revealed that compared to native species, percent nitrogen was significantly lower in the invader (Going & Dudley, 2008).

Disturbance is often cited as one of the leading facilitators of exotic plant invasions (Eschtruth & Battles, 2009). Invasion has been associated with increases in bare ground (which is similar to the greenhouse pot experiment described here) caused by disturbance and consequent increases in available light (Perry & Galatowitsch, 2006). In many instances, particularly in communities vulnerable to invasion following disturbance, light availability may be equal to or more important than nutrient availability in deciding the likelihood of invasion (Perry & Galatowitsch, 2006). Some studies suggest that plant height may play a role in invasive species' success (Wang et al., 2021). This, combined with the trend of higher relative growth rate in invasive species (James & Drenovsky, 2007), indicates that early and continued access to sunlight may allow invasive species to attenuate light more successfully than shorter, slower-growing plants in the same area. In a study of Solidago canadensis and Conyza canadensis, it was found that the invasiveness of these species was influenced mainly by plant height and, thus, higher competitiveness for sunlight acquisition. Plant height was found to be a more significant contributor to overall invasiveness than leaf photosynthetic area, especially under heavy invasion (Wang et al., 2021).

We investigated the response of A. donax to varying levels of light, moisture, and nutrients in a greenhouse experiment using plants propagated from rhizomes. Based on past performance in field and greenhouse studies investigating one or two of these factors, we made five main predictions for the response of A. donax to these treatment combinations which were as follows: (1) A. donax will have higher values for performance-related traits—traits that directly influence the functionality and fitness of an individual—such as height and photosynthetic rate, when grown under high nutrient, saturated soils without shading; (2) A. donax will have higher photosynthetic rate and lower nitrogen use efficiency (NUE), phosphorus use efficiency (PUE) and WUE when grown in saturated, high-nutrient soils; (3) A. donax will have lower overall growth when grown in the shade; (4) A. donax will exhibit growth-related responses indicating nutrient colimitation with N and P; (5) A. donax will allocate more biomass to stem tissues as opposed to leaf and rhizome tissues when grown in high nutrient, saturated soils without shading.

2 METHODS

We conducted a 4 × 3 × 2 factorial experiment of nutrients, soil moisture and light availability to better understand how these factors may interact to affect the growth potential of A. donax. This experiment investigated nutrient colimitation of nitrogen and phosphorus at four levels, soil moisture at three levels, and shading at two levels, with five replicates of each treatment (n = 120).

Plants were grown in 22.7-L (5-gallon) pots filled with commercially available locally sourced topsoil (~2.5% organic matter) and sand at a 1-part sand to three parts topsoil without any additional fertiliser. Arundo donax rhizomes were harvested on 5 March and 30 March 2021, from the Blanco Shoals Natural Area in San Marcos, Texas. Due to the genetic homogeneity among A. donax populations (Ahmad et al., 2008), one site was used for the collection of all A. donax rhizomes; therefore, site was not considered as a factor in the experiment. Rhizomes were washed, had their roots trimmed and cut into fragments weighing 20–90 g with at least one bud present (n = 150). Fragments were buried 5 cm apart and 3 cm deep in 57.15 × 40 × 12.7 cm³ tubs containing moist soil (1:3 sand to topsoil) and allowed to germinate until sprouting was observed (4–15 days). Once most fragments sprouted, wet weight was recorded, and sprouted fragments were transferred to experimental pots. One fragment per pot was buried ~3 cm deep in the soil. Plants were grown in the Texas State University Research Greenhouse for 116 days.

One week after A. donax planting, fertiliser treatments were administered at four levels, control (0 g m⁻² y⁻¹ of nitrogen and phosphorous), nitrogen addition as ammonium nitrate (34.5% N; 15 gN m⁻² y⁻¹), phosphorous addition as triple superphosphate (46% P₂O₅; 15 gP m⁻² y⁻¹), and nitrogen + phosphorous addition (15 gP m⁻² y⁻¹ + 15 gN m⁻² y⁻¹). This treatment regime follows procedures used by Smith and Slater (2010). Fertiliser was dissolved in 25 mL of water and applied to the base of the plant with a syringe. In a review of N fertilisation rates in the current literature for A. donax L., Panicum virgatum L., and Miscanthus spp., all members of Poaceae, N fertilisation rates in a variety of environmental conditions were found to range most commonly from 0 to 30 gN m⁻² y⁻¹ (Monti et al., 2019).

Moisture levels for pots were maintained at three different levels, dry (watered every third day with 350 mL of water, ~25% soil moisture), wet (watered every other day with 350 mL of water, ~32% soil moisture) and saturated (pot placed in a tray of standing water and refilled when water levels drop below 2 cm, ~40% soil moisture). Soil moisture was monitored using a Stevens HydraProbe attached to a PP Systems EGM5.

Five shade tents representing dense canopy cover in riparian areas in central Texas (Crawford et al., 2020) with an average reduction in PAR of 80%, measuring 114 × 152 × 165 cm³, were constructed using PVC and shade cloth and randomly placed across three greenhouse benches. One cohort (12 pots) of each of the five replicates of each nutrient and moisture treatment combination (60 pots total) were placed under the shade cloth after being allowed to establish in experimental pots for 1 week, while another cohort of each treatment combination were left unshaded. Pots were assigned a stratified random position on the greenhouse benches. Every other week, the plants were relocated to a new position in the greenhouse using a stratified random placement in which shaded and non-shaded sub-units (cohorts) remained grouped to minimise the effects of temperature and light gradients within the greenhouse.

3 RESULTS

3.1 Morphological traits

3.1.1 AGB and BGB

In full sun treatments, averaged across nutrient levels, A. donax's AGB increased with soil moisture, while AGB did not vary across treatments grown in shade (2-way interaction – light × moisture, p < 0.0001, Table 1 and Figure 1). Plants grown in saturated soils had greater AGB than those grown in dry and wet treatments (main effect of moisture, p < 0.0001, Table 1, and Figure 1). Plants grown in full sun had greater AGB than those grown in shade treatments (main effect of light, p < 0.0001, Table 1, and Figure 1). Similar to AGB, in full sun treatments, averaged across nutrient levels, A. donax's BGB increased with soil moisture, while BGB did not vary across treatments grown in shade (2-way interaction – light × moisture, p < 0.0001, Table 1, and Figure 1). Plants grown in saturated soils had greater BGB than those grown in dry and wet treatments (main effect of moisture, p < 0.0001, Table 1, and Figure 1). Plants grown in full sun had greater BGB than those grown in shade treatments (main effect of light, p < 0.0001, Table 1, and Figure 1).

TABLE 1. F-values and significance levels for general linear mixed-effects models using initial rhizome mass as a covariant and cohort as a random effect for the effect of nutrients, soil moisture, shade, nutrients, and soil moisture (N:M), nutrients and shade (N:S), soil moisture and shade (M:S), and nutrients, soil moisture, and shade (N:M:S) on aboveground biomass (AGB), belowground biomass (BGB), shoot:root ratio, stem:leaf ratio, average tiller length, and net photosynthesis.

Source	AGB	BGB	Shoot:root	Stem:leaf	Average tiller length	Net photosynthesis
Nutrients	3.38	0.01	8.28**	4.11**	0.53	0.31
Moisture	54.61***	100.03***	5.81*	48.28***	7.65**	51.17***
Shade	152.04***	271.05***	0.20	17.91**	0.37	2.14
N:M	0.07	0.39	0.13	1.01	0.54	0.17
N:S	0.08	0.29	0.07	0.70	0.22	1.49
M:S	50.07***	100.75***	2.25	71.01***	52.27***	28.63***
N:M:S	0.23	0.00	0.89	2.55	1.29	1.10

• * p < 0.05;
• ** p < 0.01;
• *** p ≤ 0.001.

3.1.2 Shoot:root ratio and stem:leaf ratio

Plants grown in soil with added nitrogen and phosphorus tended to have greater shoot:root ratio than those grown in soils without nutrients (main effect of nutrients, p < 0.01, Table 1, and Figure 2), while plants grown in saturated soil tended to have greater shoot:root ratio than those grown in dry and wet treatments (main effect of moisture, p < 0.05, Table 1, and Figure 2). Arundo donax's stem:leaf ratio increased with soil moisture in high light, while stem:leaf ratio did not vary across treatments grown in shade treatments across nutrient levels (2-way interaction – light × moisture, p < 0.0001, Table 1, and Figure 2). Plants grown in soil with added nitrogen and phosphorus had a greater stem:leaf ratio than those grown in soil without nutrients (main effect of nutrients, p < 0.05, Table 1, and Figure 2). Plants grown in saturated soil had greater stem:leaf ratio than those grown in dry or wet treatments (main effect of moisture, p < 0.0001, Table 1, and Figure 2).

3.1.3 Average tiller height

In full sun treatments, across nutrient levels, A. donax's average tiller height increased with soil moisture. This increase in average tiller height was not seen across treatments grown in shade (2-way interaction – light × moisture, p < 0.0001, Table 1 and Figure 3). Plants grown in saturated soils had greater average tiller height than those grown in dry or wet treatments (main effect of moisture, p < 0.01, Table 1 and Figure 3).

3.1.4 Multivariate analysis of biomass allocation

In full sun treatments, across nutrient levels, A. donax allocated its biomass to the four organ types differently depending on if it was grown in saturated or dry soils (2-way interaction – light × moisture, p < 0.001 and Table S2). Arundo donax grown in shade allocated biomass to different organs than it did when grown in light (main effect of light, p < 0.001 and Table S2). Plants grown in saturated soils tended to allocate biomass differently than plants grown in dry soils (main effect of moisture, p < 0.001 and Table S2).

3.1.5 Percent leaf tissue

In full sun treatments, across nutrient levels, A. donax allocated less biomass to leaf tissue in saturated treatments than in dry or wet treatments. Leaf biomass allocation did not vary across treatments grown in shade (2-way interaction – light × moisture, p < 0.0001, Table S2 and Figure S2). Plants grown in shade tended to allocate more biomass to leaf tissue than those grown in full sun (main effect of light, p < 0.05, Table S2 and Figure S2). Plants grown in saturated conditions allocated less biomass to leaf tissue than in dry or wet treatments (main effect of moisture, p < 0.05, Table S2 and Figure S2).

3.1.6 Percent stem tissue

Across nutrient levels in full sun, A. donax tended to allocate more biomass to stem tissue in saturated treatments than in dry or wet treatments. Stem biomass allocation did not vary across treatments grown in shade (2-way interaction – light × moisture, p < 0.0001, Table S2 and Figure S2). Plants grown in shade allocated less biomass to stem tissue than those grown in full sun (main effect of light, p < 0.01, Table S2 and Figure S2). Plants grown in saturated conditions were observed to allocate more biomass to stem tissue than in dry or wet treatments (main effect soil moisture, p < 0.0001, Table S2 and Figure S2). Arundo donax grown in pots with added nitrogen and phosphorus allocated more biomass to stem tissue than those grown in control treatments (main effect nutrients, p < 0.05, Table S2 and Figure S2).

3.1.7 Percent rhizome tissue

In full sunlight treatments, across nutrient levels, A. donax allocated less biomass to rhizome tissue in saturated treatments than in dry or wet treatments (2-way interaction – light × moisture, p < 0.05, Table S2 and Figure S2). Arundo donax grown in shade allocated more biomass to rhizome tissue than plants grown in full sun (main effect light, p < 0.0001, Table S2 and Figure S2). Plants grown in saturated conditions tended to allocate less biomass to rhizome tissue than in dry or wet treatments (main effect moisture, p < 0.01, Table S2 and Figure S2).

3.1.8 Percent root tissue

Arundo donax tended to, in full sunlight treatments and across nutrient levels, allocate more biomass to root tissue in saturated treatments than in dry or wet treatments, while root biomass allocation did not vary across treatments grown in shade (2-way interaction – light × moisture, p < 0.05, Table S2 and Figure S2). Arundo donax grown in shade allocated less biomass to root tissue than plants grown in full sun (main effect light, p < 0.0001, Table S2 and Figure S2).

3.2 Physiological traits

3.2.1 Net photosynthesis

Arundo donax's net photosynthesis in full sunlight treatments averaged across nutrient levels increased with soil moisture, while net photosynthesis did not vary across treatments grown in shade (2-way interaction – light × moisture, p < 0.0001, Table 1 and Figure 4). Plants grown in saturated soils exhibited greater net photosynthesis than those grown in dry and wet treatments (main effect of moisture, p < 0.001, Table 1 and Figure 4).

3.2.2 Nitrogen, phosphorus and WUE

Plants grown in saturated conditions exhibited higher NUE than those grown in dry conditions (main effect of moisture, p < 0.001 and Figure S3). Plants grown in treatments with added nitrogen exhibited lower NUE than those grown without added nutrients and those grown with the addition of phosphorus or the combination of nitrogen and phosphorus (main effect of nutrients, p < 0.05 and Figure S3).

Plants grown in saturated conditions exhibited lower PUE than those grown in dry conditions (main effect of moisture, p < 0.001 and Figure S3). Plants grown in treatments with added nitrogen and phosphorous exhibited no difference in PUE than those grown without added nutrients (Figure S3).

WUE of A. donax increased with soil moisture, while WUE did not vary across treatments grown in shade and tended to be higher than the full sun treatment (2-way interaction – light × moisture, p = 0.0013 and Figure S3). Plants grown in saturated soils tended to have greater WUE than those grown in dry and wet treatments (p = 0.001, main effect of moisture and Figure S3).

4 DISCUSSION

Our experiment aimed to investigate how light, nutrients, soil moisture, and their interactions affect the performance-related traits and resource allocation patterns of A. donax. Variation in light and soil moisture significantly influenced the performance-related traits of A. donax; however, nutrients were not found to significantly influence these traits in any of the treatment combinations. We did not find evidence to support our first prediction that there would be significant three-way interactions between nutrients, soil moisture, and light. However, average tiller height, AGB, BGB, net photosynthesis, and stem:leaf ratio showed a significant interaction between soil moisture and light. This suggests that tests of these resources independently may not fully explain how A. donax would respond in the field where these environmental factors vary. For example, when averaged across nutrient treatments, AGB increased with higher soil moisture in full sun conditions, while AGB remained consistent across treatment combinations in low light conditions. This indicates that soil moisture can have varying effects on AGB depending on the light conditions. Therefore, in disturbed sites where light competition has been eliminated and soil moisture is high (e.g., recently scoured riverbanks or riparian areas), A. donax will likely have a higher probability to successfully establish and become dominant. However, if the area remains shaded (woody canopy), A. donax will likely not maintain as much of a competitive advantage regardless of other environmental conditions (Quinn & Holt, 2009). Further investigation is necessary to support this hypothesis in the field.

In a model of Phalaris arundinacea in restored sedge meadows, it was found that canopy cover may lead to Phalaris arundinacea being outcompeted by native species if (1) the native species have lower light requirements than the invasive species and (2) the success of the invasive species is a result of quick establishment and competitive resource acquisition (Perry & Galatowitsch, 2006). In a separate study, shading meant to represent riparian canopy cover suppressed the establishment of another invasive member of Poaceae, Urochloa arrecta. Given the substantial negative response of A. donax to shading, and the interactions present between shading and soil moisture, it is likely that restoration of native canopy cover may provide some defence against invasion.

Research on other invasive species has indicated that the height and overall size of a species could play a crucial role in its ability to be invasive. In a review of 18 multispecies comparative studies identifying plant traits associated with invasiveness, height was identified as one of the factors that was universally associated with invasiveness (Pyšek & Richardson, 2007). This corresponds to simulation modelling results suggesting size can greatly influence the ability of a species to successfully invade (Goldberg et al., 2017). Therefore, gaining a better understanding of the factors that increase height and biomass production in A. donax, a species that can grow up to 10 m in height, is important. Previous studies of A. donax have shown that its growth and biomass production is closely related to soil moisture (Nackley et al., 2014; Quinn et al., 2007). Although capable of withstanding extreme drought and excessive soil moisture, A. donax seems to perform best in well-drained saturated soils (Perdue, 1958), which was observed in this experiment. Arundo donax has also been found to withstand the effects of intermittent shading for over 2 years (Spencer, 2012). Though tolerant of shading, the effects have proven detrimental to the ability of A. donax to accumulate biomass (Lambert et al., 2014); results of our study were consistent with these findings as both AGB and BGB were lower in shaded treatments. While independent effects of soil moisture and shading have been reported for A. donax, no studies prior to this study have reported a significant interaction between light availability and soil moisture.

Analysis of whole plant NUE showed that plants grown in saturated soils had higher NUE than those grown in dry soils. In addition, plants grown in treatments with added nitrogen exhibited lower NUE than those grown without added nitrogen except when combined with phosphorus. This suggests that A. donax uses nitrogen most efficiently when adequate water is available and that as nitrogen availability increases NUE decreases. A similar pattern was found in Triticum aestivum L. cultivars where increased soil moisture increased NUE and increased N lowered NUE (Gauer et al., 1992).

The highest photosynthetic rate occurred under full sun and saturated conditions. Drought stress is considered one of the most influential factors limiting photosynthetic rate—moderate leaf water deficits (relative water content down 70%) can greatly diminish photosynthesis in plants under drought stress (Giardi et al., 1996). Nutrients were not found to significantly influence photosynthetic rate, which was surprising given that nitrogen influenced NUE and past studies have found a correlation between N addition and an increase photosynthetic machinery (Martina & von Ende, 2012).

Although nutrient addition was not found to significantly interact with light and soil moisture for performance-related characteristics, it did influence biomass allocation in A. donax. The linear mixed-effects model found that plants with added nutrients had higher shoot:root ratio and stem:leaf ratio indicating that higher nutrient availability led to increased resource allocation for stem growth and light capture. This is further supported by our MANOVA results. We found that plants grown in soils where nutrients were added had a higher proportion of biomass allocated to stem and root tissue than plants grown in control pots, specifically those with N and P added allocated the most biomass to stem and root tissue. We did not detect any interactions between nutrient addition and other independent variables; however, a significant interaction between soil moisture and light availability was observed for the biomass allocation of all plant organs. Plants grown in full sun and saturated conditions tended to allocate less biomass to leaf and rhizome tissue and more to stem and root tissue. This tradeoff likely confers A. donax a competitive advantage over other plant species allowing it to obtain resources by gaining dominance both belowground and aboveground rapidly, a theory supported by Quinn et al. (2007). A study comparing two smaller statured mixed prairie invasive grass species—Poa pratensis and Bromus inermis—and two native grass species—Pascopyrum smithii and Stipa viridula—found that the invaders tended to allocate more biomass to roots while natives allocated more biomass to the crown (Dong et al., 2014). Invasive species using high relative growth rates and increasing traits associated with resource acquisition is not uncommon and is often what is assumed to give invasive species a competitive edge over their native counterparts.

Arundo donax grown in CO₂ and N enriched environments was found to allocate 50% more biomass to rhizomes (Nackley et al., 2017). Understanding the growth and allocation strategies of individual invasive species under varying environmental conditions can help develop an understanding of what ecosystems may be at the highest risk of invasion and under what conditions. Our experiment suggests that nutrient availability is not as important for the growth of A. donax as other resources. This knowledge allows managers to prioritise sites with high soil moisture and open canopy as our results suggest A. donax is much more successful under these conditions.

FUNDING INFORMATION

This work was supported by startup funds provided by Texas State University.

CONFLICT OF INTEREST STATEMENT

The authors have no relevant financial or non-financial interests to disclose.