Biol Invasions DOI 10.1007/s10530-016-1231-1

ORIGINAL PAPER

Response of an invasive , , to nitrogen addition: a test of form-preference uptake

Chaohe Huangfu . Huiyan Li . Xinwei Chen . Hongmei Liu . Hui Wang . Dianlin Yang

Received: 29 August 2015 / Accepted: 9 July 2016 Ó Springer International Publishing Switzerland 2016

15 ? Abstract differ in their capacity to use higher N–NH4 recovery across biomass compo- various forms of nitrogen (N). Although previous nents than both co-occurring native plants, Amaran- studies have suggested invasive plants alter N avail- thus retroflexus and Eclipta prostrata. F. bidentis ability, few distinguish their responses to various demonstrated a strong preference for ammonium ? - forms and different concentrations of inorganic N. In (NH4 ) over nitrate (NO3 ) and captured at least 15 ? order to understand how plant preference for N affects twice the N–NH4 as the native plants. By compar- invasions, we tested the growth and physiological ison, the two native species showed no preferences for response of Flaveria bidentis, an invasive plant across the form of N. The greater above-ground biomass of F. north China, to different forms and concentrations of bidentis contributed to its higher 15N recovery. We inorganic N. Seedlings of F. bidentis were cultivated suggest that the ability of F. bidentis to respond in a mothproof screen house to determine if this rapidly to changes in the N pool, especially in invader benefits from increased or altered forms of N. ammonium, may confer a competitive advantage to 15 ? - N-labeled NH4 and NO3 were applied to the soil this species over native species. Our results provide to gain insight into N partitioning in communities insight into how species-specific N preferences influ- invaded by this species. We determined that plant ence the ability of this species to invade a native growth and biomass variables, chlorophyll content, community. and photosynthesis parameters all varied depending on the form of available N. Specifically, N addition Keywords Ammonium Á Biomass allocation Á altered the biomass allocation pattern of plants, with F. Invasive plant Á Nitrate Á N preference Á Stable isotopes bidentis tending to maximize its reproductive output under increased N availability. Also, F. bidentis had

Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10530-016-1231-1) contains supple- Plant invasion, serving as a driver of global environ- mentary material, which is available to authorized users. mental change, threatens native biodiversity in many C. Huangfu Á H. Li Á X. Chen Á H. Liu Á areas, leading to profound changes in ecosystem H. Wang Á D. Yang (&) processes and function (Vila` et al. 2011), and Agro-Environmental Protection Institute, Ministry of community structure (Callaway et al. 2005; Hierro Agriculture, 31 Fukang Road, Nankai District, Tianjin 300191, China et al. 2005; Yelenik et al. 2004). Plant species differ in e-mail: [email protected] their ability to take up soil nutrients, and nutrient 123 C. Huangfu et al. mining by invasive plants can affect ecosystem introduced to many countries in , Asia, nutrient cycling (Ehrenfeld 2003). Variations in the Australia, Central and North America and Europe effects of plants on and their responses to nutrient (Gao et al. 2004). In China, F. bidentis was first cycling can result in dynamic feedbacks that influence found in 2001 in the suburbs of Tianjin and a few plant community composition and contributes to cities of Hebei Province (Liu 2005). It invades a invader persistence; however, few studies have wide range of habitats, such as roadsides, abandoned focused on these resource-based feedback mecha- fields or crop fields, outcompeting natural vegetation nisms (Bever et al. 2010). and forming dense, nearly monospecific stands Resource acquisition is one of the main ways that (Huangfu et al. 2011). F. bidentis tolerates environ- invasive species affect invaded communities. Some mental stress from salinity and cold temperature, and invasive plants can increase soil nitrogen (N) pools interferes with the development of sustainable by two orders of magnitude (Corbin and D’Antonio agriculture (Gao et al. 2004). Some evidence shows ? 2004; Vitousek and Walker 1989), thus altering the that elevated levels of N, especially NH4 , are competitive balance in favor of fast-growing inva- associated with F. bidentis invasions in agricultural sive species. Other plants affect N cycling by soils (Zhang et al. 2010). In a recent field study, the altering the ratio of soil N forms through litter presence of F. bidentis resulted in a decreased decomposition and uptake (Aanderud and Bledsoe potential nitrification rate relative to co-occurring 2009). If invasive species use nutrient resources native species in sites in both Jinghai county, Tianjin - more efficiently than native species, this may and Hengshui city, Hebei, where soil NO3 pools change resource flows and create feedbacks affect- were significantly depleted compared with unin- ? ing community biodiversity and ecosystem function vaded sites. In contrast, NH4 pools in invaded soils (Funk and Vitousek 2007; Gross et al. 2005; Pickart were generally stable or elevated (Zhao et al. 2015). et al. 1998). Nutrient acquisition strategies may Because invaded habitats are generally N deficient include details of nutrient uptake, mycorrhizal (Zhao et al. 2015), there is much interest in associations, nutrient requirements, and chemical understanding how the persistence of F. bidentis form preferences, among others. Species’ prefer- alters soil N cycling. It is unknown if F. bidentis ences for specific chemical forms of N can influence responds differently to added N forms, and the the coexistence and distribution of species within degree to which F. bidentis productivity is enhanced some sites (Andersen and Turner 2013). Invasive in the presence of specific N forms relative to co- plants tend to increase nitrification rates (Liao et al. occurring native species is poorly understood. 2008). However, little is known about the role of Soil N is particularly important in sustaining plant changes in N form in driving plant function (e.g., growth and regulating photosynthesis (Vitousek and species-specific N form-preference) or how it affects Farrington 1997). In this study, we first compared the ability of different plants to assimilate nitrate growth and biomass variables along with photosyn- - ? (NO3 ) versus ammonium (NH4 ) (de Graaf et al. thetic parameters of F. bidentis in response to N 1998; Olsson and Falkengren-Grerup 2000). Inva- addition of different forms and concentration (includ- sive species that can monopolize soil nutrient pools ing N deficiency). In the second experiment, using 15N will directly or indirectly suppress native species tracers, two native co-occurring species, Amaranthus

(Ehrenfeld 2003). If these invasive species are retroflexus L. (C4 plant, Amaranthaceae) and Eclipta susceptible to increases in total N availability or prostrata (L.) L. (C3 plant, ), were added to have different affinities for specific N forms, this experimentally quantify the acquisition of chemical type of positive feedback may further promote their forms of N. We measured recovery of 15N-ammonium 15 ? 15 15 - invasion or accelerate the spread of existing invasive ( NH4 ) and N-nitrate ( NO3 ) by the invader and species. by these two native species. We were specifically A newly introduced non-native invasive weed, interested in determining whether F. bidentis showed a

Flaveria bidentis (L.) Kuntze (C4 annual herb, preference for one chemical form of N when compared Asteraceae), commonly called ‘‘yellowtop’’, is with the native plant species and in determining if this increasingly prevalent in northern China. This preference induced shifts in biomass allocation and species originated in and has been physiological variables. 123 Response of an invasive plant, Flaveria bidentis, to nitrogen addition

Materials and methods australis, Artemisia scoparia and Artemisia annua. After cover vegetation was removed, the top 10 cm of Seedling establishment soil was collected and then passed through a 2 cm sieve to remove stones and coarse woody debris. Forest topsoil Seeds of three species, A. retroflexus, E. prostrata, and was used to provide a substrate with a natural supply of F. bidentis were collected from a site located on the macro- and micro-nutrients, and to increase soil perme- north shore of Tuanbo Lake (38°54.40N, 117°8.460E) ability in the experimental pots. We used field soil rather Jinghai County, Tianjin, China, from September– than sand culture to empirically mimic the conditions October, 2010. Climate at this site is temperate/warm that the test species experienced in the same climatic temperate, with a mean temperature of 11.8 °C. region. Before the treatments, the physical and chemical Annual precipitation is approximately 582 mm, fall- properties of the soil in pots were determined, and were ing primarily from June to August. Bulk seed collec- as follows: pH (7.2), total organic C (28.25 g kg-1), total -1 - -1 ? tions were made from 10 to 30 plants for each species; N (1.85 g kg ), NO3 –N (26.82 mg kg )andNH4 – seeds were cleaned and dry-stored in darkness at room N (4.36 mg kg-1). This potting soil N level falls within temperature (ca. 20 °C) until sowing. F. bidentis the range of N for various habitats previously invaded by produces small, water-dispersed seeds with a thousand F. bidentis (Zhao et al. 2015;Tuetal.2013). grain weight of 204.2 mg, which germinate in the field Next, seeds of the test species were sown at depths from May–October (Zhang et al. 2010). Laboratory of 0–2 cm depending on seed size. Seeds were misted seed germination trials indicated that this species is twice daily to maintain soil humidity at field capacity light-demanding, with a very low germination rate gravimetrically once a day at the germination stage. (0 %) under dark conditions (below 1 cm soil; Afterwards, all pots were watered three times weekly unpublished data). An initial germination test showed to 75 % of field capacity during the entire group of that these test species had a germination potential of experiments by weighing pots every 2–3 days and more than 70 % under laboratory conditions. The adding water based on the water loss for each pot. entire study was comprised of an initial and a follow- Each pot was watered individually using a gentle spray up experiment conducted in 2011 and 2013, respec- nozzle to minimize cross contamination by splashed tively. Genetic analysis using microsatellites has water. Pots were rearranged every other week to suggested that two Jinghai populations of F. bidentis reduce inter-pot variation during the conditioning approximately 10 km apart were genetically identical phase. Seedlings were thinned to one per pot when a (Ma et al. 2011). Therefore, the same batch of seed of sufficient number of individuals had two sets of true F. bidentis collected in 2010 was used in the following leaves to ensure that all individuals within a species two pot experiments, provided their germination were of approximately equal size (similar height and potential was still retained when sowing in 2013. basal diameter). The seedlings were grown under In late May of 2011 and 2013, seeds were sown in the ambient light, temperature and photoperiod, consis- mothproof screen house (herein, screen house) of the tent with the Tianjin location from May–August. All Agro-Environmental Protection Institute, Ministry of seedlings had achieved the eight-to-ten leaf stage Agriculture (AEPI), 21 km north of the sampling site. when experiments commenced 2 months later. The screen house was covered with a 4 cm2 square mesh net of galvanized metal to prevent damage by birds and Experiment 1 small mammals but to allow ventilation and rainfall. Identical pots (24 cm in diameter, 10 kg of soil per pot) To test whether soils with different forms and amounts were filled with equal proportions of field soil and of N had different effects on the growth and biomass commercial forest topsoil for the following two exper- allocation of F. bidentis, we conducted a pot exper- iments. Field soil was also collected from the same iment in AEPI from July to August 2011 using similar- general location where the seeds were collected, but all sized vigorous F. bidentis seedlings at the ten-leaf the test species were locally absent from the exact soil- stage. In this first experiment we maintained a control collection site to exclude possible prior species-specific and a sucrose addition treatment. The experiment was effects. Native vegetation at points where soil was set up as a randomized block design. The six collected were mainly Setaria viridis, Phragmites treatments were each replicated 16 times, resulting 123 C. Huangfu et al.

? - in 96 pots in total. NH4 and NO3 were added as production per individual plant within each treatment NH4Cl and Ca(NO3)2 salt solutions, respectively. Pots was in proportion to the biomass of buds in each were provided with 100 ml of one of six aqueous treatment. The reproductive allocations to biomass ? ? treatments: (1) low NH4 (LA), (2) high NH4 (HA), were calculated as percentage of buds biomass. - - (3) low NO3 (LN), (4) high NO3 (HN), (5) sucrose To directly address whether F. bidentis responds (C), and (6) water control (CK). All solutions were more favorably to one N form or another, in situ gas added at equivalent total N concentrations (340 and exchange measurements were made in the screen 680 mg N per pot for low and high concentration house several days before harvest on five randomly treatment, respectively). For presentation purposes in selected plants per treatment. Leaf chlorophyll content this article, the N treatments were converted to grams was measured following the procedure of Lichten- per m2, which resulted in 7.65 and 15.30 g N m-2, thaler (1987) with five replications (individual plants). respectively. The low N concentrations were selected The light-saturated photosynthetic rate (Pmax) of fully based on the quantity of mineral N observed in field expanded leaves was measured with a Li-6400 soil extractions taken from sites where this invasive portable photosynthesis system (Li-Cor, Lincoln, species occurs. Amidinothiourea (ASU), a nitrification NE, USA), using an open, steady-state gas analysis inhibitor (Shi and Norton 2000), was used in this system. Measurements were taken on one or two of the experiment to avoid possible nitrification when plants first fully expanded leaves from the apical shoot as ? were fertilized with NH4 (but see Song et al. 2015). close to their natural orientation as possible. Condi- The addition of carbon (C) to the soil as sucrose has tions in the leaf chamber were controlled automati- -1 been suggested as a countermeasure to reduce inor- cally by the equipment, with 380 lmol mol CO2, ganic N availability (Suding et al. 2004). The sucrose 60 % relative humidity, and a constant temperature of (42 % carbon) was applied as a solution with 20 g per 31 °C. Prior to the measurement, each sample leaf was pot (2 g kg-1 soil). The amount of ASU addition was illuminated with saturated light (1200 lmol m-2 s-1) calculated as 3 % of the corresponding amount of for 15 min to achieve full photosynthetic induction. applied N. Upon N fertilization, all pots were covered The light was provided automatically by the LED light with a thin layer of soil as a mitigation strategy to source of the Li-6400. The measurements were always reduce the possible emission of nitrous oxide (N2O), in taken around 11:30 a.m. Water-use efficiency (WUE) - particularly with the NO3 treatments. and transpiration rate (Tr) measurements were All pots were placed in the screen house, and obtained at the same time. seedlings were watered equally each day without any additional fertilizer. We were careful to minimize any Experiment 2 potential leaching of the soil solution during watering, especially to prevent physical losses of soil N in the To determine any preferences in the uptake of each of - form of NO3 under possible high rainfall regimes. A the labeled N forms by test species, F. bidentis and two plastic dish was placed under each pot to collect native species (A. retroflexus and E. prostrata) that leachate during the experiment. The location of each often co-occur with F. bidentis, we conducted a pot was randomly rearranged each week to minimize second pot experiment in the AEPI screen houses in possible positional effects caused by differences in July 2013. Seedlings of test species were cultivated as light and temperature within the screen house. above. There were 16 replicates (each replicate being Eight weeks after treatment, plant height, primary one pot containing one seedling) per treatment for branch number, and numbers of inflorescences were each species. Three species crossed with three N measured. Plants were then harvested and were treatments were distributed in a randomized complete separated into leaves, buds, support organs, and roots block design (3 species 9 3 N treatments 9 16 repli- for each individual plant. Roots were rinsed with cates = 144 pots). Seedlings were allowed ample time distilled water, and all plant components were dried at to adjust and 15N was added to the pot soils when all 80 °C for 48 h and weighed. The buds biomass of each test species were in vegetative growth at the eight to individual plant was calculated and served as a proxy ten leaf age, with similar plant size within the same test for seed production, rather than counting seeds and species. Each pot received 40 ml of 15N solution with risking seed dissemination. We assumed that seed equivalent concentrations of the individual N forms 123 Response of an invasive plant, Flaveria bidentis, to nitrogen addition

(99 at %, 200 lgNml-1 or 8 mg 15N pot-1)as Available N determination 15 15 NH4Cl or K NO3 (Isotec Sigma-Aldrich Co., St. - - Louis, MO, USA). The pH of the solutions was We sampled available NO3 –N and NH4 –N in pot adjusted with NaOH to 7.2, corresponding to the pH soil after harvest in both experiments. To avoid measured in soil water solution. Solutions were influencing the measurement of plant root biomass, injected evenly into the soil with an auto-refilling we did not sample pot soils before the final harvest. syringe using a 10-cm-long stainless steel needle. We collected three 1.9 cm diameter, 10 cm deep There were eight injection points around each fresh soil cores from each pot in August of 2011 seedling, and solutions were evenly delivered over a (harvest time, after removing roots) in Experiment 1. 4–10 cm depth below the soil surface to maximize The three soil cores taken from each pot were mixed root contact. One treatment contained deionized water to form a composite sample. Soil samples for 15 - - without N tracer and was used to assess the natural available NO3 –N and NH4 –N were also collected variation in d15N values among species. ASU was also after harvest in 2013 in Experiment 2 as above. ? - ? used in this experiment to retain N as NH4 in the While the resulting NO3 –N and NH4 –N concen- 15 NH4Cl treatment. The amount of ASU added was trations were strongly affected by plant uptake as calculated as 3 % of the corresponding amount of well as fertilization rate, and did not represent the applied N, and this was injected into the soil in overall quantity of N available to plants, their 15 combination with the NH4–N fertilizer after it had comparisons among treatments can provide a qual- been dissolved. The pots were watered and random- itative measure of their effects on plant-available N, ized as described for Experiment 1. for the specific times when sampled. We extracted 15 - - -1 Seventy-two hours following N application, plants NO3 –N and NH4 –N with 2 mol l KCl; soil were harvested and dried as described above. After mineral N concentrations were measured with a being ball milled to a fine powder in an Oscillating Mill flow-injection auto analyzer (Bran ? Luebbe AA3). (MM400; Retsch, Dusseldorf, Germany), plant tissues, including stems, roots, and leaves were analyzed for % Data analysis N and 15N/14N at the Institute of Genetics and Physiology, Hebei Academy of Agriculture and Experiment 1 Forestry Sciences, Hebei province, China. Powdered materials (approximately 2 mg of plant tissues) were All data were tested to determine if they met the analyzed in duplicate for total N concentration and normality assumption with a Shapiro–Wilk test, and isotopic signature by an elemental analyzer (EA, Flash we evaluated each variable for the best distribution of 2000 HT, Thermo Scientific, Waltham, MA, USA) expected versus actual residuals to identify the best coupled with a Continuous Flow Isotope Ratio Mass way to transform the data. The growth response Spectrometer (CF-IRMS, Delta V Advantage, Thermo variables (plant height, PH; primary branch number, Scientific). Nitrogen derived from fertilizer (as %) was PBN; number of inflorescences, NOI) and raw values calculated as in Hood (2001). We employed two of biomass components (root biomass, RB; support methods of presenting N uptake data among species. biomass, SB; blade biomass, BLB; total biomass, TB) We first compared uptake by computing the proportion were log-transformed and the buds biomass raw value of 15N in the biomass components of each species (BB) was square-root transformed before analysis. relative to the total 15N amount applied in the soil. To These variables were first analyzed using two-way ? determine the absolute difference in uptake of two analysis of variance (ANOVA) with N form (NH4 - forms of N among test species, we also computed the versus NO3 ) and concentration (high versus low recovery percentage of 15N in each form of N in the concentration) as the main factors analyzed without biomass components of each species relative to the the carbon addition (C) and no fertilizer (water, CK) amount of 15N added. Given the substantially higher treatments. Tukey’s HSD post hoc test was used to biomass of F. bidentis when compared to that of the determine the significance of the main factors and their two native species, we also compared dry biomass interaction. A two-way ANOVA indicated that water- accumulation and percent N by individual plants under use efficiency (WUE) was not significantly affected by the three different treatments by each species. any treatments (data not shown); therefore, this 123 C. Huangfu et al. variable was excluded from the following analysis. Results The effects of combinations of different N concentra- tions and forms on these growth variables and biomass Experiment 1 components of F. bidentis plants was tested using a one-way ANOVA with N treatment as the independent Effect of N addition on growth and reproductive variable; the means of treatments were then compared variables using Tukey’s studentized range test. Data on biomass reproductive allocations, chlorophyll content, and gas Final destructive measures of plants revealed that the exchange parameters (Pmax and Tr) were also sub- high N treatment significantly increased growth, and jected to a one-way ANOVA without prior that the magnitude of increase differed between the N ? transformation. forms (Fig. 1). In general, plants in the NH4 treat- ments were significantly larger compared with those in - Experiment 2 the NO3 treatments (Fig. 1) and all growth variables were highest under the HA treatment, especially

Plant characteristics (dry biomass and percent N) were blade biomass (BLB, Fform = 17.61, P \ 0.0001, analyzed using a one-way ANOVA for each species Fconcentration = 28.82, P \ 0.0001, Fform 9 concentration separately, with the N treatments as the main factor. = 6.036, P \ 0.01, df = 1,64), total biomass (TB, Means were separated using Tukey’s studentized Fform = 24.85, P \ 0.0001, Fconcentration = 60.85, 15 range test. Also, differences in amounts of N derived P \ 0.0001, Fform 9 concentration = 9.508, P \ 0.0001, from each fertilizer source (NdFF %) within plant df = 1,64), plant height (PH, Fig. 1a), and number of components (roots, stems or leaves) were also ana- inflorescences (NOI, Fig. 1c). For instance, plants in the lyzed using a one-way ANOVA. Natural logarithm HA treatment accumulated 133.6 % more total biomass transformations of the data were performed as needed than those in the CK treatment (water control), how- to meet assumptions of homoscedasticity prior to ever, plants in the LN treatment accumulated only ANOVA. Preferences in the chemical form of 15N 75.4 % of the total biomass of those in the CK treatment taken up by different species were tested by paired t- (Fig. 1d). Further, a significant increase in floral tests when normality and homogeneity of variance production (160 and 413 % increase for NOI and BB, assumptions were met, either directly or after natural respectively, P \ 0.05, Fig. 1c, d) was observed in the logarithm transformation; all data in figures are back- HA treatment compared with the CK treatment. Due to transformed values. Total % 15N recovery as well as this form-specific response, the increments of most factors and explanatory variables were incorporated variables in the HN and LA treatments were approx- into an analysis of covariance (ANCOVA) model, imately equal (P [ 0.05). Also, primary branch number which used backwards elimination as a model selec- (PBN) was lowest in the LN treatment (P \ 0.05, tion technique. In this part of the analysis, 15N form Fig. 1b). During 2 months after fertilization, C addition treatments and species were used as independent resulted in a decreased growth trend in F. bidentis variables and the plant components (roots, stems and compared with the control, with significant differences leaves) as covariates to specifically test if these detected for PBN and NOI (P \ 0.05, Fig. 1b, c). variables contribute to 15N recovery across species. In general, the reproductive response of F. bidentis Because plant biomass may have differed between 15N was significantly affected by all treatments except for form treatments and species by the end of the the C addition compared with the CK treatment. First, experiment, natural logarithm transformed dry bio- there was a significant increase in reproductive mass data was included in the analysis. In this way, the allocation at the individual plant level with the effects of the main factors on % 15N recovery could be increasing N concentration when compared with the evaluated by comparing plants of a similar biomass. CK treatment. This increase reached the greatest in the The interaction between 15N form treatments and HN and LA treatments (P \ 0.05, Fig. 2), although species was included in the analysis. the reproductive allocation was not significantly Alpha was set at P = 0.05 for all analysis, and different among the HN, LA and HA treatments. In SPSS for Windows with Version of 17.0 (SPSS Inc., addition, no significant difference was observed Chicago, IL, USA) was used. between the HA and LN treatments (P [ 0.05, Fig. 2). 123 Response of an invasive plant, Flaveria bidentis, to nitrogen addition

b 120 (A) Form 13.514* Fig. 1 Average growth response variables, plant height (a), Conc 226.310*** a primary branch number (b), number of inflorescences (c) and b b 100 Form Conc 4.517** c dry biomass components (d) for F. bidentis across different fertilization treatments. Values are mean ± SE (n = 16). 80 d Different lowercase letters denote significant differences among d 60 treatments for the same variable based on one-way ANOVAs, while different capital letters denote significant differences 40 among treatments in total biomass at P = 0.05. F values above

Plant height (cm) 20 bars of each variable show significant differences for main effects and their interaction. N form (Form), N concentration 0 (Conc), Form 9 Conc indicate F-statistic, *P \ 0.05; CKC LNHNLAHA **P \ 0.01; ***P \ 0.001. CK water control, C sucrose, LN - - ? ? Treatment low NO3 , HN high NO3 , LA low NH4 , HA high NH4 , RB root biomass, SB support biomass, BLB blade biomass, BB buds 16 biomass (B) Form 32.660*** a a 14 Conc 72.169*** 12 Form Conc 19.833*** a

10 b 25 8 c 6 a c 20 a 4 ab 2 15 b Primary branch number 0 bc CK C LN HN LA HA 10 c Treatment 5 180 a Form 63.159*** 160 (C) Reproductive allocation (%) 0 Conc 245.023*** b CK C LN HN LA HA 140 Form Conc 32.901*** c 120 Treatment 100 Fig. 2 Biomass reproductive allocation (%) of F. bidentis at the 80 d whole plant level under the six fertilization treatments. Values 60 d e are mean ± SE (n = 16). Bars followed by the same letters are 40 not significantly different according to Tukey’s HSD test at 20 - Number of inflorescences P = 0.05. CK water control, C sucrose, LN low NO3 , HN high 0 NO -, LA low NH ?, HA high NH ? CKC LNHNLAHA 3 4 4 Treatment

30 A (Table 1). Chlorophyll content, Pmax and Tr were (D) BB BLB SB RB a lowest under the C and CK treatment as predicted 25 )

1 AB - B (Table 1). F. bidentis plants that received any con- a 20 a a centration of N showed greater chlorophyll content b 15 b and photosynthesis capability, except for the LN C b C 10 b a treatment. Chlorophyll content and gas exchange bc a C b c a capability were highest in the HA treatment compared c 5 b b b ab with all other treatments (P \ 0.05). bc c c ab a Dry biomass (g plant 0 One-way ANOVA indicated significant differences CK C LN HN LA HA (P \ 0.01) between N treatments in NH ? and NO - -5 Treatment 4 3 levels at harvest time. Soils in the NH4Cl treatment ? - tended to have higher NH4 –N and lower NO3 –N - Effect of N addition on physiological variables concentrations, and lower NO3 inorganic N ratios than other treatments (P \ 0.01, ESM 1), showing that Chlorophyll content and photosynthesis parameters the use of a nitrification inhibitor was effective. Also, for F. bidentis across different N addition treatments C addition resulted in significantly decreased inor- exhibited a similar trend to the growth response above ganic N pools (P \ 0.01, ESM 1), suggesting that our

123 C. Huangfu et al.

use of the C treatment to mimic N deficiency 0.23a 0.79a water conditions was appropriate. 0.12a in 2011 ± ± ± CK Experiment 2 F. bidentis

st. N form (Form), N Species characteristics 0.91ab 21.06 0.65b 11.04 0.10ab 2.23 ± 5), and means labeled with ± ± transpiration rate, F. bidentis exhibited greater plant size than either A. = Tr

n retroflexus or E. prostrata, but not all the treatments had an effect on biomass production within each SE (

± species (P [ 0.05, Fig. 3a). All three test species had 0.19bc 9.61 0.92bc 19.09 0.11bc 2.06 similar percentages of plant N (from 3.99 to 5.93 %); ± ± ± no significant differences were detected in plant percent N across N form treatments when tests for each species were conducted separately (P [ 0.05, Fig. 3b). The total inorganic N concentrations of all 0.04cd 8.83 0.60c 17.50 0.06c 1.77 soils at harvest did not differ across plant treatments ± ± ± (P [ 0.05, ESM 1), potentially because of the short light-saturated photosynthetic rate, duration of the experiment (72 h). Also, the amounts

max 15

P of added N-labled were negligible compared with the inorganic N pool in the soil. 0.05). 0.21d 8.05 0.47bc 14.68 0.04bc 1.58 = ± ± ± (A) 45 0.001. Values in the rest columns are mean P a a 40 15 15

) CK NH Cl K NO

\ a 4 3 -1

P 35 30 25 ? 4 0.19cd 7.21 0.81c 17.02 0.07c 1.81 20 0.01; *** ± ± ± 15 \

P 10 a high NH a a Dry biomass (g plant 5 a a a

HA 0 ,

? Fb Ar Ep 0.05; ** ), concentration (low and high) and their interaction on dependent physiological variables for 4 - Conc CK C LN HN LA HA 3 \ Species 9 P 7

low NH a a (B) a a a a and NO 6 ? LA 4 ,

- 5 a 3 a a -statistics; *

F 4 3 high NO

Percent N (%) 2 HN ,

- 1 3 Conc indicate

9 0 N form N Conc Form 1,19 1,19 1,19 21.429*** 33.773*** 7.569** 7.89 14.347*** 8.593*** 4.893** 15.57 Fb Ar Ep ) 17.373*** 7.320*** 5.792** 1.62 low NO 1 Species - seedlings at the ten-leaf stage were fertilized and grown for 8 weeks in a mothproof screen house in Tianjin, China before determination s LN 2

- Fig. 3 Dry biomass (a), percent N (b) per plant among each

2 15 15 species within 72 h of applying either NH4Cl or K NO3 Om 2 )

2 fertilizer. Values are mean ± SE (n = 16). Different letters sucrose, ) Effects of different inorganic N form (NH - F. bidentis 1

C indicate significant differences between treatments for each - mol CO s l

( plant component at P = 0.05. CK = deionized water without 2

- 15 (mmol H N tracer; Fb = Flaveria bidentis;Ar= Amaranthus retro- values in the first three columns show significant differences for main factors and their interaction tested by two-way ANOVA followed by Tukey’s HSD te (mg dm m max df Chlorophyll content P Tr control, Table 1 Identical F Conc (concentration), Form the same letter are not significantly different from other means based on one-way ANOVAs ( flexus;Ep= Eclipta prostrata 123 Response of an invasive plant, Flaveria bidentis, to nitrogen addition

15 N recovery (A) 5.0 a 4.5 15 4.0 For F. bidentis, we recovered more N when supplied 3.5 a 15 ? 15 - as N–NH4 than when supplied as N–NO3 , 3.0 regardless of the plant biomass components studied. 2.5 2.0 b For instance, roots of F. bidentis recovered over twice b NDFF (%) 1.5 15 ? 15 - a as much N–NH4 (4.14 %) than N–NO3 1.0 b (1.64 %, Fig. 4a). F. bidentis derived a greater 0.5 15 15 ? 15 0.0 percentage of N (in both N–NH4 and N– Roots Stems Leaves - NO3 form) from the soils than the two native species. 15 Recovery of N by plant biomass components of the (B) 2.5 native species, A. retroflexus and E. prostrata, was a 2.0 generally not affected by the form of applied N a b a (Figs. 4b, c) with the exception of roots of A. 1.5 retroflexus and leaves of E. prostrata. Recovery of a 15 15 - 1.0 N by roots of A. retroflexus from N–NO3 15 ? NDFF (%) a (2.05 %) was higher than from N–NH4 (1.24 %, 0.5 P \ 0.05, Fig. 4b), while leaves of E. prostrata 15 - captured 1.10 times more N–NO3 (0.143 %) than 0.0 15 ? Roots Stems Leaves N–NH4 (0.068 %, P \ 0.05, Fig. 4c). 15 When N recovery of different N forms was (C) 1.2 compared among species, F. bidentis had the highest a 15 ? 1.0 N–NH4 recovery across biomass components a compared with either A. retroflexus or E. prostrata 0.8 a a (P \ 0.05, Fig. 5a). The roots, stems and leaves of F. 0.6 bidentis recovered 4.43 (stems) to 9.56 (leaves) times

15 ? NDFF (%) 0.4 more N–NH4 (P \ 0.05) than the respective com- a ponents of E. prostrata. Meanwhile, A. retroflexus 0.2 b displayed similar differences in the recovery of this N 15 ? 0.0 form by capturing at least 1.39 times more N–NH4 Roots Stems Leaves 15 - than E. prostrata (P \ 0.05). For N–NO3 , stems and roots of F. bidentis captured more 15N–NO - than Fig. 4 Comparisons of percent N derived from two N form 3 fertilizer (NDFF, %) by plant components in three test species those of E. prostrata (P \ 0.05, Fig. 5b). No differ- Flaveria bidentis (Fb, a), Amaranthus retroflexus (Ar, b) and 15 - 15 ences were observed in N–NO3 recovery in stems Eclipta prostrata (Ep, c) within 72 h of applying either NH4Cl 15 F. bidentis A. retroflexus (black bars)orK NO3 (white bars) fertilizer. Values are between and , but leaves of 15 the latter recovered more 15N–NO - than that of the mean ± SE (n = 16). Differences between N form treatments 3 are shown (paired t test, P = 0.05) invader (P \ 0.05). In general, when compared with both native species, F. bidentis had higher recovery of 15 both N forms present in all plant biomass components, explaining the variation in N recovery at the species with this species deriving 7.77 and 3.27 % N at the level. This model incorporated the main factors and 15 ? 15 - their interaction (test species, 15N form, species 9 15N whole plant level from N–NH4 and N–NO3 applied in soils, respectively. The recovered 15N form) and plant dry biomasses (roots, stems and 15 accounted for no more than 2.75 % in the two native leaves). Our total % N recovery was predominately species. influenced by test species (F = 71.46, P \ 0.0001), followed by the N form (F = 58.23, P \ 0.0001) and ANCOVA model their interaction (F = 26.36, P \ 0.0001). The differ- ence in biomasses of both stems (F = 14.80, An ANCOVA backwards elimination regression P = 0.001) and leaves (F = 10.45, P = 0.003) were model was used to determine factors and/or variables significant. 123 C. Huangfu et al.

(A) 5.0 a Reynolds et al. 2003). However, a recent meta- 4.5 Ep Ar Fb analysis by Ordonez and Olff (2013) suggested that 4.0 these benefits may remain the same for both native and 3.5 a non-native species, and that the effects of soil nutrients 3.0 on invader performance may vary, and they at least

(%) 2.5 depend on the resource availability conditions at a 2.0 particular site (e.g., Gurevitch et al. 2008). Recent b

NDFF b 1.5 studies have emphasized the importance of N form on c 1.0 a changes driving shifts in plant communities; a favor- c b 0.5 able response to one form of N over another may help c 0.0 to explain the dominance of many plants (Andersen Roots Stems Leaves and Turner 2013; Cui and Song 2007; Daehler 2003; Davis et al. 2000). Examination of how invaders differ 1.8 (B) a from natives in N uptake and use, in addition to below- 1.6 a ground nutrient dynamics, may thus be important for 1.4 b understanding shifts in soil N availability. Invasive 1.2 a species may differ from native species in their use or 1.0 c affinity for a particular form of N or its concentration 0.8 a in the soil (Fraterrigo et al. 2011; Hewins and Hyatt b NDFF (%) 0.6 b 2010; Ross et al. 2011). For example, the invasive 0.4 plant Microstegium vimineum can alter soil microbial c 0.2 communities or nutrient availability, thereby provid- 0.0 ing more suitable conditions for conspecific individ- Roots Stems Leaves uals; its productivity was highest in monocultures - receiving NO3 ; in contrast, uninvaded native com- Fig. 5 Comparisons of percent N derived from fertilizer munities showed no response to N form (Lee et al. (NDFF, %) among three test species divided by plant 15 2012). components within 72 h of applying either NH4Cl (a)or 15 K NO3 (b). Values are mean ± SE (n = 16). Differences Invasive plants associated with altered soil nutrient among test species are shown by different letters (one-way availability often have physiological and morpholog- ANOVA, P = 0.05). Ar = Amaranthus retroflexus;Ep= ical traits that enhance N acquisition and N-use Eclipta prostrata;Fb= Flaveria bidentis efficiency (Ehrenfeld 2003). Plant traits (e.g., growth rate, biomass allocation) are often associated with the Discussion response of plants to changing N quantities and ? qualities. In general, NH4 –N addition had a greater Enhanced soil inorganic N levels have often been overall effect on F. bidentis than the two native species observed with invasive plants (e.g., Adair and Burke tested in these experiments. When N concentration 2010; Hellmann et al. 2011; Rout and Chrzanowski was increased, F. bidentis tended to allocate greater 2009) and the response of invasive plants to N has biomass to plant reproductive components (reproduc- often been tested in numerous manipulative field and tive allocation). However, F. bidentis biomass did not greenhouse experiments. These studies conclude that - ? respond more strongly to NO3 than to NH4 ,aswe invasive plants are more generally favored by expected. The growth responses of F. bidentis in the increased N availability and responded more rapidly HN treatment were similar to those in the LA to N availability than native species (e.g., Brooks treatment for most variables measured (Fig. 1). 2003; Lowe et al. 2003; Sun et al. 2009). Accordingly, ? Greater overall growth of F. bidentis in the NH4 the presence of invasive plants in a community might ? treatments suggested that NH4 –N is the N form that lead to positive feedback when both the causes and influences invasion success and that this non-native benefits from altered soil N are considered, helping the species does not respond similarly to different chem- species to maintain its dominance in the invaded ical forms of N inputs. F. bidentis was more produc- community (Bever et al. 2010; Ehrenfeld et al. 2005; tive and showed superior photosynthetic parameters in 123 Response of an invasive plant, Flaveria bidentis, to nitrogen addition

? - NH4 - than in NO3 -dominated soil conditions species (Blumenthal et al. 2003). C source addition ? (Fig. 1; Table 1), suggesting that NH4 -dominated increases soil fungal and bacterial populations which soils may confer F. bidentis a competitive advantage use decomposing organic matter as their energy source over the native community. and immobilize available N from the soil, making less Although F. bidentis responded strongly to N available to plants (Blumenthal et al. 2003; Perry increased N concentrations, it was still able to survive et al. 2010; Wilson and Gerry 1995). Decreased N, in and reproduce even in the C addition treatment turn, might reduce growth of invasive plants (mostly (Fig. 1), especially for buds biomass (BB). Almost nitrophilic), thereby releasing native species from every individual in the experiment was able to produce competitive suppression. In a meta-analysis, Hauben- an inflorescence even when growth in height and sak et al. (2007) concluded that C addition decreases biomass were limited (i.e., C addition treatments) as the success of invasive plants, but some studies even Ross et al. (2011) found with M. vimineum.This found that native species were favored under these finding indicates that F. bidentis might be more plastic conditions (e.g., Blumenthal et al. 2003). In a recent relative to the native species used here in its response review of 50 studies, Perry et al. (2010) found that C to both N forms at different concentrations. In terms of addition, along with other methods (burning, grazing, photosynthesis, for example, the C addition treatment biomass removal, topsoil removal) used to lower soil had no effect on F. bidentis in respect to chlorophyll N availability with the goal of establishing desirable content, Pmax and Tr (Table 1), regardless of the species tolerant of low-N conditions was the most higher allocation of fixed carbohydrate reserves to promising approach in managing plant invasion to floral production. Aside from N, other factors such as date. Based on our results, we suggest that C addition light and competition may be more important factors might be one way to inhibit the growth of F. bidentis in in determining seed production (Claridge and Franklin practice. Because of its rapid but short-lived effect,

2002). As a C4 annual herb that is adapted to high light sugar is often used in combination with other more environments and has high N-use efficiency, F. complex C sources such as sawdust to provide both bidentis can persist in a wide range of habitats (Zhao rapid and long-term C pools based on large scale et al. 2015), as long as adequate light is available application (Bleier and Jackson 2007; Haubensak et al. (Claridge and Franklin 2002). If non-native invasive 2007). species can more efficiently convert available N into Resource partitioning or niche differentiation of biomass than native species, they will be more likely limiting resources may allow different species to co- to overgrow and/or outcompete neighboring native occur in this ecosystem (McKane et al. 2002; Song species (Bobbink et al. 1998). This ability may allow et al. 2015). Partitioning could be spatial (rooting for their spread and establishment in additional, varied depth), temporal (seasons) and/or dependent on N environments. Because plant growth is an allometric form. We conducted the 15N-tracer experiment to process, any factor that affects plant size might also compare the uptake of different chemical forms of N influence plant reproductive allocation. Data related to by both native and invasive plants to determine if whole-plant allometric reproductive allocation sug- preferences for N forms changed as a result of 15 ? gests that more biomass was allocated to the repro- invasion. F. bidentis recovered more N–NH4 than ductive organs than to vegetative ones. This also the native species in this study, and similar trends were indicates that F. bidentis would maximize its repro- observed in species-specific N preferences for 15N– ductive output under increasing N availability, thus NO3. Among biomass components, roots of F. bidentis maximizing the risk of it spreading via seeds. captured more 15N than those of the other species, Increases in N availability can favor fast growing suggesting the importance of N form in creating invasive species over slow growing native species. niches. The heightened plasticity of this species may Therefore, techniques that reduce the availability of also apply to N foraging preferences. Preferences for ? - soil N to the plant community might restore the NH4 over NO3 were exhibited by the plant biomass competitive balance in favor of natives. C addition components of F. bidentis, but no consistent prefer- (so-called ‘‘reverse fertilization’’, Hunt et al. 1988) has ences were observed for the native species. Acquiring ? - been suggested as a method for immobilizing plant- NH4 is less energetically costly than NO3 in uptake available N and increasing the success of native and assimilation, constituting an advantage for plants 123 C. Huangfu et al.

? that are very competitive for NH4 absorption instance, biomass NUE was estimated to be ca. 31 % (Boudsocq et al. 2012). Instead of preferring the most higher in invasive M. vimineum-dominated plant com- dominant N form, F. bidentis might have greater munities than in diverse native wetland plant commu- ? potential for use of NH4 , indicating its flexibility in nities (DeMeester and Richter 2010). With higher NUE, terms of source foraging. We inhibited nitrification of C4 plants can allocate more N to the production of ? the added NH4 , thereby ruling out the possibility that structures associated with the increased capture of the - test species took up NO3 that had been converted resources that mostly limit overall plant growth, and ? from NH4 . The niche partitioning of N form among have a significant fitness advantage over C3 plants in co-occurring species, combined with the capability of N-limited environments when light is not limiting. In F. bidentis to recover higher amounts of specific N this present study, this variation in biomass was even forms, might contribute to the faster growth rate of this evident between two natives, A. retroflexus (C4 plant species relative to the native species (Aanderud et al. with biomass of 3.774–4.188 g/plant) and E. prostrata

2003). These two results might help explain not only (C3 plant with biomass of 0.397–0.684 g/plant). Even the ability of F. bidentis to invade a broad range of though no differences in percent N content or dry soils in northern China but also could explain the wide biomass within each species among the treatments was variability of habitats during the invasion process observed, a great difference in biomass accumulation throughout the region. The persistence of A. retro- was observed between F. bidentis and two natives in flexus and E. prostrata in F. bidentis-invaded sites Experiment 2. This finding suggests that this invasive might be partially attributed to their niche differenti- species is able to gain a greater size and therefore more ation of N form uptake. competitive ability with less N use. Consequently, F. ? - The discriminate uptake of NH4 and NO3 , bidentis and other invasive species with C4 photosyn- ? especially when NH4 is preferred, would lead to thesis might have a significant fitness advantage over C3 rhizosphere soil acidification (Arnold 1992). This was plants in N-limited environments when other nutrient supported by our previous studies showing that the pH sources are equal. of F. bidentis rhizosphere soil decreased compared Based on the ANCOVA backwards elimination with non-invaded soils (Zhang et al. 2010; Zhao et al. regression model, total % 15N recovery was predomi- - 2015). Furthermore, NO3 synergistically promotes nately influenced by species followed by N form. The the uptake of cations, such as K?,Ca?, and Mg?, significant interaction between species and N form ? while excess NH4 reduces the availability of these highlights the presence and importance of N form cations for plants because of their different charges; preferences in determining soil N recovery even after therefore, co-occurring species suffer N toxicity and accounting for the differences among species. Biomass growth inhibition (Hoffmann et al. 2007; von Wire´n of both stems and leaves can influence total 15N et al. 2000). recovery, while the difference in root biomass among In a recent study, Fraterrigo et al. (2011) added 15N species did not limit 15N capture. Thus, the greater tracers to natural invasions of M. vimineum and reported biomass, especially in stems and leaves, of F. bidentis that this species allocated most N to above-ground when compared with the two native species contributed biomass. Although we found higher biomass accumu- to its higher 15N recovery. It is possible that this lation with F. bidentis (28.384–29.235 g/plant) than additional allocation of N above-ground will shift N with the native species, no difference in percent N from below- to above-ground food webs in an F. between above- and below-ground biomass across N bidentis-invaded ecosystem (Fraterrigo et al. 2011). forms was detected (data not shown). Greater amounts Accounting for the fact of higher above-ground biomass of biomass can be mainly explained by high N-use of F. bidentis and that above- and below-ground litter efficiency (NUE, ratio of photosynthetic rate to N inputs are thought to be under different controls in investment in the leaf, DeMeester and Richter 2010). affecting ecosystem N cycling (Jo et al. 2016), we High NUE under favorable (i.e., high light) conditions suggest that F. bidentis invasion could alter invaded is a general characteristic of C4 photosynthetic plants, community decomposition dynamics mainly through which require three to six times less Rubisco (an N-rich above-ground litter inputs, and that environments with a ? enzyme) to achieve similar or higher photosynthetic greater relative abundance of NH4 –N may play a larger rates than C3 plants (Ehleringer and Monson 1993). For role in aiding its above-ground competition. 123 Response of an invasive plant, Flaveria bidentis, to nitrogen addition

Our results offer a case insight into how species- microbes (Bardgett et al. 2003; Owen and Jones specific N preferences may influence plant invasions. 2001). Neither glycine nor free amino acid concen- ? The N preference of F. bidentis for NH4 might have trations have been measured at our research site. contributed to its rapid invasion. However, N metabo- However, a recent study on the types and amounts of lism can also change with plant age as suggested by amino acids present in soils of different ecological Hewins and Hyatt (2010). For example, Allium sp. system in China has also indicated that free amino acid biomass in the early stages of growth is maximized content was lower in amount among the extractable or- ? - when NH4 is the sole source of N, but NO3 is required ganic N pool, particularly in arable soils (Li et al. by older plants (Abbe`setal.1995). In Vigna and 2002). Therefore, we assume the pool of amino acids - Glycine sp., NO3 uptake increases throughout vege- is small and the uptake of amino acids was quantita- - tative growth, peaks when the reproductive stage tively less important than the uptake of NO3 and ? begins, and decreases during seed formation (Imsande NH4 at this site. Nevertheless, McKane et al. (2002) and Edwards 1988; Imsande and Touraine 1994). The proposed that in situations where organic N use by ecological consequences of such preferences will plant species is common, the potential exists for niche influence the distribution of plants and biological partitioning between species based on the chemical diversity at a variety of scales. Thus, to better under- form. Additional experiments and bioassays of the stand the whole picture, a need exists to focus our future uptake of amino acids by F. bidentis would strengthen work on studying whether the invader can gain any our understanding of N acquisition and the resource extra benefit from the available N shift, especially in the use strategies of this invasive plant. presence of interspecific competition from co-occurring Nevertheless, our present results suggest that either local species (e.g., Lee et al. 2012). Our previous studies the form or the concentration of N can explain the have indicated that the invasion of F. bidentis affects relative success of F. bidentis over native species in the ? - nitrification rates but the direction and magnitude field. Increased N availability, as either NH4 or NO3 varies, depending on the location of the specific or both, can promote invasion by species such as F. research sites (Zhao et al. 2015). For instance, F. bidentis or Alliaria petiolata (Hewins and Hyatt 2010). - bidentis soils had significantly reduced NO3 –N in Because of their ability to take up multiple forms of N Jinghai and Hengshui compared with uninvaded soils, and respond vigorously to increases in N supply, both of - while a significantly increased amount of NO3 –N was these non-native invasive species pose major chal- available in Xian County. This trend was also demon- lenges to land managers working with already highly strated by changes to the potential nitrification rate invaded habitats. Since deposition of N is increasing in (Zhao et al. 2015). F. bidentis grows in a wide variety of northern China because of fossil fuel use and increased habitats, including wasteland that we investigated in the agricultural application of N (Liu et al. 2011), this effect present study, and on roadsides and even in arable fields will be further magnified and non-native invasive (Tu et al. 2013). Different experimental sites or species such as F. bidentis will continue to spread. methodology employed could influence conclusions, and research on N uptake related to M. vimineum,for Acknowledgments This work was funded by the Natural instance, yielded inconsistent results, from no prefer- Science Foundation of Tianjin (12JCQNJC09800), the Special Fund for Agro-scientific Research in the Public Interest ence for inorganic N (Fraterrigo et al. 2011;Rossetal. (200803022, 201103027), National Natural Science - 2011)toNO3 –N preference (Lee et al. 2012). Foundation of China (No. 31401811) and National Key Consequently, studies in other parts of the invaded Research and Development Program (2016YFC1202200). range of F. bidentis are needed to evaluate whether the This article is dedicated to the memory of my beloved sister, ? - QY, Huangfu. Former master candidates from our lab, including preference on NH4 over NO3 is common to this NN, Wang and XH, Zhao, provided lab and common garden invasive species. assistance. The authors would also like to thank three Dissolved organic N (i.e., amino acids) has been anonymous reviewers for providing excellent comments and considered to be a non-significant N source for plant critical suggestions that greatly improve this manuscript. growth, mainly because of its typical low concentra- Compliance with ethical standards tion in soil solution (Christou et al. 2006; Jones et al. 2005; Kielland 1994; Raab et al. 1999) and low Conflict of interest The authors declare that they have no competitiveness in use by plants relative to soil conflict of interest. 123 C. Huangfu et al.

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