Nitrogen uptake kinetics and saltmarsh plant responses to global change

Coastal wetlands are important carbon sinks globally, but their ability to store carbon hinges on their nitrogen (N) supply and N uptake dynamics of dominant plant species. In terrestrial ecosystems, uptake of nitrate (NO3−) and ammonium (NH4+) through roots can strongly influence N acquisition rates and their responses to environmental factors such as rising atmospheric CO2 and eutrophication. We examined the 15N uptake kinetics of three dominant plant species in North American coastal wetlands (Spartina patens, C4 grass; Phragmites australis, C3 grass; Schoenoplectus americanus, C3 sedge) under ambient and elevated CO2 conditions. We further related our results to the productivity response of these species in two long-term field experiments. S. patens had the greatest uptake rates for NO3− and NH4+ under ambient conditions, suggesting that N uptake kinetics may underlie its strong productivity response to N in the field. Elevated CO2 increased NH4+ and NO3− uptake rates for S. patens, but had negative effects on NO3− uptake rates in P. australis and no effects on S. americanus. We suggest that N uptake kinetics may explain differences in plant community composition in coastal wetlands and that CO2-induced shifts, in combination with N proliferation, could alter ecosystem-scale productivity patterns of saltmarshes globally.

have only been investigated in a small number of studies, making it difficult to generalize how different species or functional groups may adjust N uptake in response to elevated CO 2 .
Although photosynthetic pathways typically determine plant physiological responses to elevated CO 2 , the circumstances under which these physiological differences translate into a change in N uptake kinetics is not yet clear. While N acquisition is generally not affected by elevated CO 2 in C 4 plants, C 3 plants show variable patterns in uptake parameters under elevated CO 2 (e.g., V max or K m ) 9 . For example, in studies of temperate forest trees, the effect of elevated CO 2 on NH 4 + root uptake capacity was species dependent, ranging from +215% in Acer negundo to −40% in Quercus macrocarpa 33 . In related studies, elevated CO 2 increased the maximum rate of NO 3 − uptake, specifically in Pinus ponderosa, Bouteloua eriopoda and Pinus taeda [34][35][36][37] . Other studies have found no significant effect of elevated CO 2 on NO 3 − or NH 4 + uptake rates, namely in Pinus taeda, Prosopis glandulosa, Ceratonia siliqua, and several herbaceous species [35][36][37][38][39][40] . Furthermore, effects of elevated CO 2 are not limited to N uptake rates. In the case of a C 3 tropical seagrass, Halodule uninervis, elevated CO 2 inhibited NO 3 − assimilation and NO 3 − nutrition alone did not enhance the CO 2 response 41 . One proposed explanation for the species-specific effects of elevated CO 2 on NO 3 − assimilation in C 3 plants is that elevated CO 2 concentrations decrease photorespiration, thereby decreasing the amount of reductant (NADH) available to support NO 3 − reduction to NO 2 − in the first step of NO 3 − assimilation 42 . In contrast, the C 4 carbon fixation pathway generates sufficient quantities of reductants in the cytoplasm of mesophyll cells, thus avoiding the inhibitive effect of elevated CO 2 on NO 3 − assimilation 43 . The physiological capacity for nitrogen uptake and assimilation may provide a key mechanistic explanation for interspecific differences in sensitivity to CO 2 and N addition 9 . For example, N uptake dynamics may explain why N addition can favor coastal wetland species that do not respond strongly to elevated CO 2 (i.e., Spartina patens), ultimately negating the enhanced productivity response at the ecosystem-level 10 . In the context of anthropogenically-induced changes to the carbon and nitrogen cycles in wetland ecosystems, information on physiological responses of N uptake to elevated CO 2 could be highly relevant for understanding these species shifts and how they influence critical ecosystem-level phenomena such as resilience to sea level rise and carbon sequestration 44 .
Functional taxonomic groups are often linked with suites of traits, allowing for an extrapolation of results beyond a particular ecosystem. Herbaceous-dominated systems such as grasslands, deserts, tundra, and marshes are often dominated by distinct functional groups (e.g., C 3 grasses), and traits associated with these functional groups may influence ecosystem-scale responses, such as shifts in net primary productivity and carbon sequestration, to interacting global change factors 10 . Coastal saltmarshes in North America are typically dominated by C 4 grasses (e.g., Spartina patens (Aiton) Muhl.) or C 3 sedges (e.g., Schoenoplectus americanus (Pers.) Volkart ex Schinz & R. Keller). However, an introduced lineage of the C 3 grass Phragmites australis (Cav.) Trin. ex Steud. (common reed) is invading coastal and other wetlands throughout North America 45 , likely altering their response to global change 22 .
We investigated the N uptake kinetics of three functionally distinct foundation plant species in North American coastal wetlands under ambient and elevated CO 2 conditions, and related these results to the growth of each species in response to global change factors with data from long-term in situ experiments. Specifically, we asked three questions: (1). Does N uptake capacity differ between Phragmites australis, Schoenoplectus americanus, and Spartina patens? Plants adapted to low nutrient environments typically invest considerably in belowground organs and consequently do not have a high maximum uptake capacity 46 . Given that S. patens invests in belowground organs to a lesser extent than P. australis and S. americanus, we predicted that it would have a higher maximum uptake capacity. (2). Does elevated CO 2 affect N uptake kinetics? Given the decline in tissue N status observed in many plants grown under elevated CO 2 despite adequate N supply, we predicted that elevated CO 2 would negatively affect the uptake kinetics of NO 3 − and NH 4 + in all three species. (3). Do N uptake kinetics of our species explain observations in the field? We hypothesized that patterns in N uptake kinetics would correspond to species' productivity responses to both CO 2 and N in long-term field experiments.

Results
Kinetics of NO 3 − and NH 4 + uptake. We performed a series of 15 N uptake assays to test the hypothesis that saltmarsh species from contrasting functional groups (C 4 grasses, C 3 sedges, and C 3 grasses) would differ in their N uptake characteristics. In a semi-controlled outdoor setting, we presented clonally propagated plants with varying concentrations of either 15 NO 3 − or 15 NH 4 + and measured rates of N uptake by their root systems. All three species showed curvilinear relationships between N uptake rate (V uptake ) and N concentration that closely adhered to Michaelis-Menten reaction kinetics (Fig. 1). Moreover, all three species exhibited substantially greater V uptake for NH 4 + than NO 3 − , with rates differing by up to a factor of 10. There were interspecific differences in V uptake for both NH 4 + and NO 3 − (Table 1). S. patens was primarily responsible for these differences, as it exhibited mean uptake rates up to 3 times greater than those of P. australis or S. americanus (Fig. 1a,c) and separated from both species in pairwise comparisons ( Table 2). In addition, for NH 4 + , S. americanus had 20-30% greater mean V uptake across the range of N concentrations than did P. australis (Fig. 1a). These interspecific differences also manifested in the parameter V max , the maximal uptake rate, when Michaelis-Menten curves were fit to the data; in this context, V max reflects a species' capacity for N uptake under saturating N conditions. Using bootstrapped 95% confidence intervals (CIs), we again found that S. patens had Scientific RepORts | (2018) 8:5393 | DOI:10.1038/s41598-018-23349-8 greater V max than either of the C 3 species for both NO 3 − and NH 4 + (Fig. 2a,b), and that S. americanus had a greater V max for NH 4 + than did P. australis (Fig. 2a). We carried out an additional set of assays under elevated CO 2 to determine how the N uptake kinetics of our focal species could shift under future atmospheric conditions. We found that elevated CO 2 altered patterns of N uptake, though these effects were species-specific and differed by N form and concentration ( both C 3 species when grown under elevated CO 2 , and the separation between S. americanus and P. australis in these metrics was maintained under elevated CO 2 ( Fig. 2a,b, Table 2). However, for NO 3 − , elevated CO 2 induced a reduction in mean V uptake for P. australis, such that it was not differentiable from S. americanus in either CO 2 setting (Fig. 1c vs. d, Table 2).
For NO 3 − , interspecific differences in V uptake depended on N concentrations and the CO 2 level (Table 2), with CO 2 inducing larger shifts within species at low N concentrations (Fig. 1c,d). Correspondingly, there was no evidence of CO 2 affecting V max in any species, whereas it induced notable shifts in the Michaelis-Menten parameter K m (Fig. 2d). In the context of this study, K m reflects a species' affinity for an N form, with smaller values indicative of greater affinity. The shift in K m under elevated CO 2 was again greatest (and statistically unequivocal) for S. patens; bootstrapped CIs did not overlap. S. patens thus had a greater affinity for NO 3 − under elevated CO 2 (Fig. 2d). Although the corresponding 95% CIs for P. australis were partly overlapping and the three-way interaction was not significant for V uptake (Table 1), our data were consistent with P. australis experiencing the opposite shift, namely a decrease in affinity (i.e., an increase in K m ) for NO 3 − under elevated CO 2 (Fig. 2d). The data were likewise statistically equivocal for S. americanus (i.e., CIs were overlapping), as were CIs for all species with respect to NH 4 + (Fig. 2c).  Table 2. Mean rates of inorganic N uptake (V uptake ; µmol g −1 h −1 ) across N concentrations by the three focal species. Group letters that differ within an N form denote statistical separation in pairwise comparisons of means.

Growth responses to global change factors.
To determine if N uptake kinetics can explain species responses to inorganic N eutrophication in the field, we compared the results of our assays with data on biomass production (aboveground and rhizome) from two long-term field experiments in which elevated CO 2 and NH 4 + were added factorially to plots in a Chesapeake Bay saltmarsh 10,22 . In the first five years of these experiments' lifespans, N enrichment positively stimulated aboveground biomass production by S. patens and P. australis (by 214 and 220 g m −2 , respectively; Fig. 3), with stimulation defined as the absolute difference in productivity between treatment and control conditions. In contrast, S. americanus responded negatively to N enrichment (−61 ± 27 g m −2 ; mean ± SE). Elevated CO 2 positively stimulated aboveground biomass production for all three species, with the inter-annual mean change being greatest for P. australis (82 ± 26 g m −2 ), intermediate for S. americanus (20 ± 7 g m −2 ), and smallest for S. patens (7 ± 1 g m −2 ; Fig. 3a). Responses to the combined treatment (elevated CO 2 + N) were likewise greatest for P. australis and S. americanus; they produced 202 and 161 g m −2 more than they did under the control, respectively (Fig. 3a). Belowground, P. australis also had the strongest growth responses to all three treatments, with the largest mean stimulation to rhizome biomass production observed under elevated CO 2 + N (445 ± 175 g m −2 , Fig. 3). N enrichment did not affect rhizome biomass stimulation of S. americanus (−10 ± 28 g m −2 ) and S. patens responded more strongly belowground to N enrichment (18 ± 7 g m −2 ) than to the other two treatments (Fig. 3).

Discussion
Our results suggest that plant responses to interacting global change factors may be related to differences in N acquisition kinetics among plant functional groups. In a prior analysis of the native saltmarsh community's response to CO 2 and N at our site, C 4 grasses respond strongly to N addition 10 , demonstrating that N-induced plant community shifts can alter the ecosystem's productivity response to elevated CO 2 . Our data suggest that this shift may be attributable to a difference in the N uptake capacity of the dominant C 3 and C 4 species in the community (S. americanus and S. patens, respectively). A high capacity for nutrient uptake, V max , is considered to be an adaptation to nutrient rich conditions, whilst a low K m denotes a high affinity for the substrate 7 . Here, V max levels for NH 4 + uptake under ambient conditions were 150% higher in S. patens than in S. americanus, indicating that it is a high-nutrient species capable of taking advantage of N enrichment (Figs 1-3). In contrast, S. americanus is a low nutrient specialist (evidenced by low V max and low K m ), and has a limited ability to take advantage of increased soil N (Fig. 3). Furthermore, plant species with a high V max generally do not produce a high root length density and are therefore competitively inferior when nutrients in soil solution are chronically low 46 . Consistent with this pattern, fine root production was, on average, twice as high in stands of S. americanus than in stands of S. patens over the past 20 years 23 . Given this, the divergent response of these two North American wetland species to elevated N is likely attributable to differences in their N uptake kinetics, and can therefore be used in a predictive framework to project plant community shifts in response to global change.
Plasticity in N uptake physiology may explain the ability of P. australis to thrive in both resource-poor and resource-rich habitats. For example, our results and those of a prior study 47 suggest that P. australis is adapted to a low N environment, given its low V max . However, intermediate V max levels have been measured in P. australis 13,48 , as have levels an order of magnitude greater than we found 49 . As suggested by Romero et al. 49 , the ammonium uptake kinetics of P. australis seem to be plastic, such that they can be modified in response to nutrient availability or CO 2 availability. The plastic response of N uptake to varying CO 2 and N levels in the field study may partly explain why introduced P. australis can thrive under both high and low nutrient environments 50 . Our results provide evidence that P. australis has the kinetic parameters needed to invade low nitrogen environments, whilst our long-term field study shows that the species can thrive in resource rich environments. Furthermore, our long-term study clearly shows that P. australis can take advantage of both CO 2 and N, with aboveground biomass stimulated most strongly by N addition, and belowground biomass stimulated most strongly by CO 2 + N (Fig. 3).
Elevated CO 2 affected saltmarsh functional groups differently. Both C 3 species (P. australis and S. americanus) exhibited a trend for lower affinity for NO 3 − under elevated CO 2 conditions, evidenced by increases in K m values. The functional group-specific effect of elevated CO 2 on NO 3 − uptake capacity corresponds to differences in NO 3 − assimilation previously reported by Bloom et al. 32,43 and may be attributable to the reduction in photorespiration that C 3 plants experience under elevated CO 2 conditions, as this decreases the reductant available to power the first step of NO 3 − assimilation 42 . Conversely, evidence of this repression was not observed in the C 4 species, for which K m values actually decreased under elevated CO 2 . Again, elevated CO 2 conditions appeared to reduce NO 3 − assimilation in C 3 , but not C 4 , plants. It is now well established that the active process involved in ion uptake by plant roots at relatively low nutrient levels (10-200 µM) is provided by the high affinity transport system (HATS) 8,51,52 . This transport system is used by plants growing in natural and semi-natural ecosystems 9,53 and is likely the one operating at the concentrations observed in our field experiment 54 . The HATS for both NO 3 − and NH 4 + is subject to regulation in response to changes in external N availability or in the N demand of the whole plant 55 . However, the mechanisms underlying the suppression of N uptake under elevated CO 2 remain unclear.
We found that elevated CO 2 enhanced the physiological capabilities of our C 4 species, such as increasing V uptake of NH 4 + . This suggests that some C 4 plants may become more competitive for N with near-future global change. Furthermore, this taxa-specific nutrient uptake response to elevated CO 2 may influence differences in growth rate, as rates of N acquisition are often positively correlated with growth rates [56][57][58] . Indeed, S. patens had the kinetic parameters of an exploitative, fast growing species 59 , and data from the long-term field experiment show that its shoot biomass response to elevated N was on par with that of the invasive species P. australis. These physiological enhancements induced by elevated CO 2 may also explain the stimulation effects of CO 2 observed in C 4 species at a 30 year experiment at our field site 23,60 . However, rapid NH 4 + uptake does not necessarily translate into rapid growth; Zerihun & BassiriRad 33 found that the relative growth rate of Acer negundo was unaffected by high CO 2 despite experiencing a two-fold increase in root NH 4 + uptake capacity in response to high CO 2 . Elevated CO 2 appeared to repress NH 4 + uptake affinity in our dominant C 3 species; this trend may help explain long-term observations in our field experiment. The increasing K m values for NH 4 + under elevated CO 2 could partly explain the reduction of tissue N levels experienced by foundational saltmarsh plants 60 . In some species, such as wheat, the reduction occurs despite the supply of high doses of nitrogen 30 indicating that the observed reduction in tissue nitrogen with elevated CO 2 is not due to a low nitrogen concentration in the root medium, but is related to aspects of uptake itself. Indeed, N addition did not sustain the initial positive CO 2 stimulation of C 3 biomass in one of our in situ experiments 10 . This was partially explained by competition with S. patens, but may also be attributable to sustained CO 2 enrichment having a gradual decreasing effect on NH 4 + uptake capacity. In addition, the combined effects of N and CO 2 on P. australis shoot biomass was smaller than the effect of N alone, suggesting a potential negative effect. Another contributing factor may indeed be the reduction of transpiration-driven mass flow of N through soils due to a reduction in stomatal conductance usually experienced by plants under elevated CO 2 conditions 29 . Alternatively, the pattern may derive from reductions in root respiration, given that greater tissue N content entails greater maintenance respiration 61 and the fact that the energy requirements for NH 4 + and NO 3 − uptake and assimilation constitute a significant portion of root respiration 62 . Reductions in root respiration as a result of elevated CO 2 exposure have been reported in the literature 63 but no satisfactory mechanisms to explain these effects have been demonstrated 64,65 .
As is the case for NO 3 − uptake, the mechanisms underlying the suppression of NH 4 + uptake under elevated CO 2 remain uncertain. What is clear is that carbon metabolites such as glutamine can suppress the expression of genes associated with the HATS for both NO 3 − and NH 4

+66-68
. Additionally, glucose supply to plant roots can inhibit the induction of some enzymatic proteins such as glutamate dehydrogenase and asparagine synthetase 69 , both of which are involved in N metabolism 70 . The fact that C and N metabolism are tightly linked is inescapable 71 , and it may be the case that increased carbohydrate supply to roots as a result of elevated CO 2 exposure may act directly or indirectly on plant nitrogen pools, ultimately causing a downregulation of genes associated with N uptake.
The extent to which N uptake is influenced by edaphic factors such as oxygenation of the rhizosphere, salinity conditions, or sulfide concentration was not investigated in this study. To evaluate uptake free of these effects, assays were conducted in aerobic solutions free from Na or hydrogen sulfide . Salinity is known to inhibit N uptake in Spartina alterniflora and P. australis by up to 40% 13,17,48 , especially at levels above 20 ppt. Similarly, anoxic conditions and hydrogen sulfide can inhibit N uptake 18,48 . How these factors influence N uptake in the field is unknown, although S. americanus has the ability to oxygenate its rhizosphere and can tolerate frequent flooding whilst S. patens inhabits higher saltier zones 72 . Therefore each species is specifically adapted to tolerate one of these confounding factors.
The interspecific differences in N uptake kinetics identified here provide an explanation for how individual plant-level responses to global change factors (such as CO 2 and N enrichment) translate into species dynamics at a community level. We suggest that the ecosystem-level response to interacting global change factors can be related to the root uptake kinetics of N acquisition by different plant functional groups. Our results further demonstrate that P. australis is capable of invading low nitrogen ecosystems, whilst our long-term field study shows that it can also thrive in resource rich environments. Consequently, physiological plasticity in the invasive species appears to facilitate its proliferation. Further study is required to determine if rising atmospheric CO 2 levels can be expected to repress N uptake in other ecosystems and to examine the specific mechanisms involved.
Scientific RepORts | (2018) 8:5393 | DOI:10.1038/s41598-018-23349-8 Methods Nitrogen uptake assays. Three wetland taxa were selected for this study: Schoenoplectus americanus, Spartina patens, and a lineage of Phragmites australis subsp. australis (haplotype M). All three are highly abundant in saltmarshes along the Atlantic Coast of North America and are representative of the plant functional groups that dominate tidal marshes, namely C 3 sedges, C 4 grasses, and C 3 grasses, respectively. P. australis subsp. australis is both introduced and invasive in North America 73 , while the two natives are dominant species in two long-term experiments situated at the Smithsonian Environmental Research Center (SERC) in Maryland, USA.
The nutrient uptake experiment was conducted in a set of six chambers (1.0 × 0.7 × 1.0 m) located at Bryn Mawr College in Pennsylvania, USA (40.0297°N, 75.3139°W). During the course of the experiment, plants experienced natural temperature fluctuations, with a mean daily high of 29.8 ± 0.8 °C and a mean daily low of 19.5 ± 0.5 °C. The chambers had closed walls constructed of Lexan polycarbonate, though they were not air-tight. Blowers continuously moved air into chambers at a rate that replaced the volume of each chamber once approximately every two minutes. Three chambers were maintained at ambient CO 2 and three at elevated CO 2 (ambient +300 ppmv CO 2 ). CO 2 concentrations in the chambers were monitored with CM-0212 CO 2 loggers (CO 2 Meter, Ormond Beach, USA) and adjusted manually on a daily basis.
Plant material was collected in the spring of 2012 from SERC, maintained for one year in the Bryn Mawr College greenhouse, and propagated from rhizome fragments or emergent shoots in May 2013. Propagules were washed clean of organic matter and dead root material, and individual shoots were placed in square pots (10 cm sides) filled with clean sand to facilitate transfer to a hydroponic medium during N uptake assays. Thirteen plants per species were placed in each chamber in June 2013 (n = 234 total plants) and fertilized weekly with a 1/10 th strength Hoaglands solution. Within 10 weeks, individual plants achieved a root mass suitable for assays (>100 mg dw).
To investigate NH 4 + and NO 3 − uptake kinetics, we presented individual plants with a 15 N-labeled substrate in hydroponic solution. The protocol for assays was adapted from Epstein et al. 74 and Mozdzer et al. 13 . Briefly, plants were washed free of sand and placed in an N-free solution of 0.50 mM CaCl 2 overnight to maintain root epidermal cell integrity. After equilibration, each plant was exposed to one of six different N concentrations (5,10,25,50,100, and 500 μΜ) of either 15 NH 4 Cl or K 15 NO 3 , respectively (99% enriched; Cambridge Isotope Laboratories, Andover, USA) for 45 minutes in a well-mixed 0.50 mM CaCl 2 solution. To ensure that drawdown would not exceed 10% of the starting concentration, the reaction volume for assays was adjusted to 2500 ml for the lowest two concentrations and 1000 ml for the remaining concentrations. The treatment assay solution was identical to the equilibration medium but contained the labeled N dose. Each exposure series for both forms of N was applied to the three species in each chamber, such that the complete set of assays was performed in triplicate (n = 216 plants). One additional plant per species from each chamber was exposed only to the equilibration medium as a control (n = 18 plants). After 45 minutes of exposure, roots were rinsed for 2 min with 1 mM KCl to remove any excess labeled substrate from root surfaces. Each plant was then separated into root, rhizome, and stem tissue and dried at 60 °C to constant weight. Dry tissue was ground using a Retsch Mixer Mill 400 (Verder Scientific, Haan, Germany). To minimize potential effects of diurnal variation in nutrient uptake, assays were conducted at approximately the same time each day (1000-1200 h) over the course of three weeks, with the three exposure series for one plant species, one N form, and one CO 2 level (n = 18 plants) completed per day. Samples of root tissue were analyzed for 15  where m 1 is the mass of N in the sample (in μg), APE samp is the atom % excess 15 N of the root sample exposed to a labeled substrate, APE ctrl is the atom % excess 15 N in the control root sample, APE treat is the atom % excess of the labeled 15 N treatment, MW is the molecular weight of the N isotope, m 2 is the dry root mass of the sample (in grams), and t exp is the duration of the exposure to labeled substrate (in minutes). Several uptake rates were anomalously high, especially at low N concentrations (5-25 μM). This was probably due to carryover during mass spectrometry, so V uptake values that were greater than those at both of the next two higher N concentrations within a series were omitted (n = 19).
The 15 N uptake rates from each exposure series (n = 14-18 plants) were then fit to the Michaelis-Menten equation in order to derive values of maximal uptake rate (V max ) and the substrate concentration at which the rate is 50% of V max (K m ): parameter estimates (via nlsBoot, also from nlstools; n = 999 iterations). Estimates were considered different if there was no overlap between pairs of bootstrapped 95% confidence intervals 76 .
Linear models were used to determine how experimental factors (CO 2 level, plant species, and N concentration) affected N uptake rates (V uptake ), with separate models fit to data for NO 3 − and NH 4 + . Both models had the same form, with species and CO 2 level included as categorical variables but N concentration included as a continuous variable. Terms for all possible two and three way interactions were also included. V uptake values were square root transformed to ensure residual normality. Tukey-adjusted pairwise comparisons were subsequently made among all species-CO 2 level combinations; the family-wise error rate was held at 0.05. All statistical analyses were conducted in R version 3.2.3. Aboveground biomass is estimated in both experiments in late July or early August of each year. For S. americanus and P. australis, this entails combining stem density counts with measurements of stem height and width that are converted to dry mass using species-specific allometric relationships 77 . For S. patens, biomass is measured directly by clipping samples within each chamber. Rhizome productivity is estimated from annual samples collected each year using ingrowth cores, with three placed in each plot for the native marsh study and six placed in each plot for the P. australis study.

Long-term field experiments.
Biomass data from the two long-term field experiments were used to quantify productivity responses to global change factors. Specifically, we calculated stimulation effects (i.e., differences from ambient) for the elevated CO 2 treatment, the N enrichment treatment, and the combination treatment for both aboveground biomass and rhizome biomass. For the native marsh study, we used biomass data from S. americanus and S. patens spanning the first five years that data were available (2006-2010 for aboveground biomass and 2007-2011 for rhizome biomass). Biomass data for P. australis came from the second study, but likewise spanning the first five years of its lifespan (2011-2015).
Data availability. The datasets used in this study are available from the corresponding author on reasonable request.