Chao Peng ·Jia Tu ·Ming Yang ·Yong Meng ·Meiqun Li ·Wensheng Ai

Abstract The community species abundance and diversity declined with bamboo invasion had been widely reported worldwide.However,the physiological strategies used during root competition between native species and invasive bamboo are poorly understood.To clarify the mechanisms underlying such strategies,the stoichiometric dynamics and homeostasis of nitrogen,phosphorus,organic carbon in root orders of Phyllostachys edulis (I du [=I-Pe,years 1 and 2];II du [=II-Pe,years 3 and 4]),Cunninghamia lanceolata in transition and pure forests were analyzed.With increasing intensity of bamboo invasion,N,P,and C content of C.lanceolata root orders declined,N and P content in P.edulis rhizome orders declined,while C increased,the stoichiometric ratios in mixed forest interface mainly increased,and the stoichiometric differences within native and invasive species root orders narrowed.Meanwhile,the stoichiometric homeostasis index (H) of elements in the same root order and even the same elements in different root orders were not consistent.H of most root orders (except some H P) was greater than 4,the H ranked order was I-Pe >Cl >II-Pe in mixed interfaces,and the N:P ratio of most species root orders was greater than 16,despite being affected by invasion.Our research concluded that the bamboo invasion narrows stoichiometric differences within root orders,and the juvenile bamboo rhizome has a stronger capacity for homeostatic regulation than in adult bamboo and C.lanceolata,which is a key determinant of bamboo invasion success.

Keywords Bamboo invasion·Root orders·Nutrient dynamics·Homeostatic regulation coefficient

Introduction

Plants obtain nutrients from the soil nutrient pool via their root systems,and the correlation between concentrations of soil nutrients is considered as an indicator of resource utilization strategies for plants in a changing environment(Ordoñez et al.2009;Hong et al.2015).Therefore,root interactions define community structure,and finally,stability (Casper et al.2003).Stable root stoichiometry is crucial for regulation of plant physiology and life cycle and for interspecific competitiveness when the environment changes(Kooijman 1995;Elser et al.2000),since higher nutrient plasticity provides plants a long-term competitive advantage (Wang et al.2012;Kramer-Walter and Laughlin 2017).Previous studies conf irm significant differences in the stoichiometric characteristics of plant nutrients among various organs due to variable functions (Xu et al.2010;Jiang et al.2017).However,plant stoichiometric characteristics are also associated with plant growth (Zeng et al.2016) and environmental changes (e.g.,invasion,drought,fertilization) (Guo et al.2014;Nie et al.2018;Zhao et al.2018).Plant stoichiometric characteristics result from long-term adaptations to the environment,which also ref lect physiological strategies optimized for heterogeneous habitats (He and Han 2010).Although belowground stoichiometric strategies have been discussed extensively (Kramer-Walter and Laughlin 2017),information on root order effects is still lacking.

Bamboo is a category of fast-growing forest plants that plays an important role in culture and economic activities in Asia.However,the gradually increasing area of bamboo forest greatly threatens neighboring plants,mainly by extension of clonal flagellate rhizomes (Ouyang et al.2016;Ying et al.2016).Bamboo shoots occupy the native forests and compete for space and soil nutrients through the growth of rhizome—root systems,a process that alters the physicochemical properties (Yang et al.2011;Fukushima et al.2015;Ying et al.2016) and microbial communities of native soil (Lin et al.2014;Xu et al.2015;Li et al.2017),and ultimately weakens community stability and ecological function (Ying et al.2016).The invasion success of bamboo is mainly attributed to its morphology (strong rhizome system),high resource utilization ability,rapid growth rate,and physiological integration (Liu et al.2013,2017).However,the interspecific interaction of underground processes of invasive and native species remains poorly understood.

Therefore,we investigated the response mechanisms of invasive and native species root systems to invasion using stoichiometric theory.The objectives of this study were (1)to clarify the trends of stoichiometric structure and dynamics of native and invasive species root orders and (2) to discuss the function of stoichiometric homeostasis in bamboo invasion.

Material and methods

Study area

Three sites with a similar hilly landform and slopes from 15°to 42° were selected on the Hongheling forest farm,Linxiang City,Mid-South China (29°27′ N,113°29′ E) (Table 1).The region is characterized by a humid subtropical monsoon climate with a frost-free period of 259 days.Average annual temperature is 16.4 °C,and the mean annual precipitation is 1469.1 mm.Forest management histories of the sites were similar,and they had been free from human interference for more than 7 years.The soil type in this region is brown earth with a high gravel ratio due to cultivation and long-term erosion,and the depth range of soil layer is 22—90 cm.The intensity and frequency of long-term forest management are relatively low due to rising labor costs in this region.

Phyllostachys edulis(Pe,moso bamboo) andCunninghamia lanceolata(Cl,Chinese fir) are the two dominant species in the study area,both of which have high economic values in southern China.Chinese fir forest stands were planted about 20 years ago,and thePepopulation is in uneven-aged stands consisting of 1—5a individuals.The dominant understory species includesRhus chinensis,Cibotium barometz,andClerodendrum cyrtophyllum.

Experimental design and plant investigation

The study was conducted from September to October 2019.A belt transect (20 m × 60 m) was established at each site across the bamboo invasion front and divided into three 20 m × 20 m quadrats,each with different forest type:pure bamboo forest,bamboo—Chinese fir mixed forest,and pure Chinese fir forest.For determining invasive periods and sampling roots,four sampling sub transects (5 m × 20 m)were established in each quadrat (Fig.1),each for different stand type:moso bamboo (PES),moso bamboo mixed with few Chinese firs (PCM),Chinese fir mixed with few moso bamboos (CPM),and Chinese fir (CLS).Bamboo age was expressed as“du”to account for the phenomenon of“onyear”production,when more than 90% of shoots are produced compared with the few shoots produced in the“off -year”(Tang et al.2016):an age of one (I)“du”forP.edulisincludes years 1 and 2 years (written as I-Pe),two du (II-Pe)represents years 3 and 4,and three du (III-Pe) corresponds to years 5 and 6 (Tang et al.2016).

Fig.1 Sampling layout of three forest stands and four sampling sub transects in each 20 m × 60 m Pe-Cl belt transect in Hongheling forest farm,south-central China.CLS,Chinese fir forest;CPM,Chinese fir forest mixed with few moso bamboos;PCM,moso bamboo forest mixed with few Chinese firs;PES,moso bamboo forest

Root sampling

In each sub transect,three individuals per species (Cl,I-Peand II-Pe) were chosen randomly.Root samples were collected from the 0—60 cm soil layer where bamboo rhizomes were mainly distributed.The sampling point was about 0.5 m away from the plant stem.Root and rhizosphere soil samples were collected using a steel root drill (10 cm diameter × 25 cm height),hand-separated from soil and then approximately 100 g biomass of each fresh root was sorted carefully into labeled valve bags.All root samples were stored in a 4 °C thermostat box and taken immediately to the laboratory,where root samples were carefully cleaned with fresh water,and species identified according to their shape,color,and smell (Finér et al.1997).The separated root samples were divided into fine (<2 mm,RO1),intermediate(2—5 mm,RO2) and thick roots (>5 mm,RO3) using vernier calipers (An et al.2017).All root samples were steamed in an oven at 105 °C for 30 min,and then dried at 60 °C until constant mass.Finally,the dried samples were ground,sieved through 100-mesh sieve,and bagged for testing.

Soil sampling

In each sub transect,soil was sampled in parallel with root systems,labeled and bagged,taken to the laboratory,and air-dried in the shade.Identifiable plant residues,stones,soil animals and root fragments were removed,and soils were ground and sieved through 2-mm and then 0.15-mm mesh,quartered and bagged for chemical analyses.

Chemical properties

Soil and root organic carbon (OC) were measured by the K2C r2O7-H2S O4digestion method.Total soil and root nitrogen (TN) were determined with the Kjeldahl method (K-370,Buchi Laboratory Equipment,Flawil,Switzerland) (Zeng et al.2016).Soil and root total P (TP) were determined using the Mo-Sbcolorimetry methodwithH2S O4-HClO4digestion(Zengetal.2016).Soilammoniumnitrogen NH4+-N(AN)was analyzed using a KCl extraction and Skalar SAN++continuous flow analyzer (Skalar,Breda,Netherlands) and available soil P (AP) was determined by the Mo-Sb colorimetry method with NaHCO3soaking (Wang et al.2015).

Statistical analyses

Before analysis,normal distributions of all variables were determined with a one-sample Kolmogorov—Smirnov test(K-S test),and a homogeneity test of variance was carried out.If homogeneity was not found,the corresponding values were log-transformed.Spearman correlationanalysis was conducted to test for correlation of C,N,and P between soil and roots.Multiple stand comparisons of soil nutrients and stoichiometric ratios in various diameter classes of roots were tested with a one-way ANOVA followed by the Duncan multiple range test when differences were significant.Homeostatic index (H) was calculated using the model proposed by Elser et al.(2000) as:

whereyis root TN,TP or corresponding N:P ratio,xis AN,AP or corresponding N:P ratio,cis constant,andHis the homeostatic index.SpeciesHvalues in the mixed interface stands,which are not the simple addition ofHin each root order,were calculated without distinguishing root orders.Statistical analyses were conducted using SPSS 21.0 software (IBM,Armonk,NY,USA).Figures were prepared using Sigmaplot 14.0 software (Systat Software,San Jose,CA,USA).

Results

Stoichiometric soil characteristics with bamboo invasion

Available nitrogen (AN) in the pure bamboo stand was significantly higher than in the other stands(PAN=0.045),with a content of 11.18 mg kg−1(Fig.2).Available phosphorus (AP) in the mixed interface stands(CPM and PCM) was significantly higher than in the pure forest stands (CLS and PES) (PAP=0.049).However,there were no significant differences in organic carbon(POC=0.106),total nitrogen (PTN=0.188),total phosphorus (PTP=0.095),and their ratios among all forest stands.

Fig.2 Changes of soil nutrient stoichiometry with bamboo invasion.CLS:Chinese fir forest;CPM:Chinese fir forest mixed with few moso bamboos;PCM:moso bamboo forest mixed with few Chinese firs;PES:moso bamboo forest;OC:soil organic carbon;T:total;A:available.Total and available nutrient units are g·kg−1 .Different letters denote significant difference at P <0.05

Stoichiometric dynamics of root orders with bamboo invasion

N and P ofClandPeroot orders changed variously under the influence of invasion (Figs.3 and 4).As the number of invasive bamboo shoots increase,N content in Chinese fir root orders decreased significantly (P<0.05) (from 11.81 g kg−1to 9.29 g kg−1),but declines in C (from 416.8 g kg−1to 410.7 g kg−1) and P (from 0.54 g kg−1to 0.47 g kg−1) content were not significant (P>0.05).In rhizomes along the direction of bamboo invasion in I-Peand II-Pe,N significantly changed (from 7.28 g kg−1to 6.20 g kg−1and from 1.08 g kg−1to 6.83 g kg−1,respectively),P decreased but insignificantly (from 0.41 g kg−1to 0.33 g kg−1,and from 0.56 g kg−1to 0.33 g kg−1),while C significantly increased(from 346.8 g kg−1to 432.38 g kg−1,and from 365.5 g kg−1to 418.0 g kg−1).The ratios C:P and C:N ofClandPeroot in pure stands (CLS and PES) were higher than those in the mixed interfaces (PCM and CPM).The N:P ratio in most root orders did not change significantly.

In pure forest stands (CLS and PES),N,P and N:P in RO1 were higher than in other orders,while C in RO1 was lower than the other orders;C:P and C:N had corresponding significant differences.However,with bamboo invasion(PCM and CPM),most differences were less obvious except for N content inClandPeand C and C:N inPe.

Correlation between soil and root nutrients during invasion

Most root N was negatively correlated with total soil N except for II-Pe(r2=0.333,P>0.05) in the PES stand(Table 2).However,root N was positively correlated with available soil N,root P was positively correlated with soil TP and negatively with soil AP,and root and soil organic C also were positively correlated in all but the CLS stand.The few significant or highly significant correlations between soil and root nutrients in various forest stands indicated that the soil nutrient pool did not significantly influence thestoichiometry of most roots during invasion periods in this region.

Table 2 Spearman’s correlation coefficients for root and soil total (T) and available (A)nitrogen,phosphorus and organic carbon (OC) without distinguishing root orders of each individual in the same homogeneous soil habitats

Fig.3 Stoichiometric Cunninghamia lanceolata root order trends with bamboo(Phyllostachys edulis) invasion.CLS,Chinese fir forest;CPM,Chinese fir forest mixed with few moso bamboos;PCM,moso bamboo forest mixed with few Chinese firs.Letters a (or A) and b (or B) denote significant difference for the same root order at different invasive periods,c (or C) and d (or D) denote significant difference for root orders of each species in the same forest stand.Capital letters denote significant difference at P <0.05,lowercase letters at P <0.01

Stoichiometric homeostasis index of root systems in forest stands

The stoichiometric homeostasis index (H) of mostClandPeroot orders was greater than 4 exceptHPin some root orders (such asCl HPof RO3 in PCM),indicating that the dominant species in the region were mainly homeostasis types (Fig.5).As bamboo invasion progressed (from CLS to PCM),HNandHPof eachClroot order showed variable patterns,HNof all orders increased (with means from 12.67 to 80.60 for orders),andHPof RO1 declined from 19.3 to 2.52,whileHPof RO2 and RO3 initially increased and then declined.Additionally,theHN:Pof all orders decreased rapidly (means from 48.35 to 8.04 for orders).On the basis of these results,Clroot orders adjusted their stoichiometric homeostasis in a feedback that affected invasion.ForPe,rhizome ordersHNandHN:Pin mixed interfaces (PCM and CPM) were higher than in PES,HNin both RO1 and RO2 initially increased,then decreased with invasion;however,HPrhizome order decreased.TheHN:PofPeRO1 order was PCM (15.87) >CPM (14.52) >PES (6.09),and the RO2 order increased along the direction of invasion.WhenHvalues of the root orders for the two species were compared,a variable response of stoichiometric homeostasis was shown among the functional root system.

Fig.5 Nutrient homeostasis indices of Phyllostachys edulis(Pe) and Cunninghamia lanceolata (Cl) root orders with invasion.CLS:Chinese fir forest;CPM:Chinese fir forest mixed with few moso bamboos;PCM:moso bamboo forest mixed with few Chinese firs;RO:root order.The hyphen above a bar denotes H <0

Comparison of stoichiometric homeostasis among species in mixed interfaces

Obvious homeostasis regularities were found among the three species in mixed interfaces (Fig.6).I-PerhizomeHvalues were significantly higher than the others,and the rank order for bothHNandHPwas I-Pe>Cl>II-Pe.Additionally,the I-PerhizomeHN:Pwas higher thanCland II-Pe,but there was no significant difference betweenCland II-Pe.Thus,I-Pe had highest stoichiometric homeostasis,which indicated that the highly stoichiometric homeostasis inPerhizomes might play a key role in its invasion.

Fig.6 Comparison of homeostasis index of species roots without distinguished orders in the mixed interface (PCM and CPM)

Discussion

Dynamic changes in root stoichiometric characteristics to bamboo invasion

Nutrient element composition is closely related to physiological response in plants,and differences in the various stoichiometric relations in the root system between native and invasive species play a key driving role in plant invasion (Ma et al.2015;Ulm et al.2017).Our results suggested that root orders of Chinese fir and moso bamboo had various stoichiometric responses to invasion (Figs.3 and 4).As invasion strengthened (bamboo shoots increased),N and P of Chinese fir root orders declined to varying degrees,with corresponding changes in the elemental ratios.Bamboo rhizome N and P were positively correlated with increasing shoots,a response also conf irmed for other invasive species(Hu et al.2016;Kramer-Walter and Laughlin 2017).Niu et al.(2007) considered that there was a positive feedback between N and P absorption and the degree of species invasion,which is consistent with our results.Both native and invasive species undergo a trade-off in their stoichiometric structures to adapt to the effects of the invasion.

Comparing the trends of species in the invasive process,the Chinese fir root stoichiometric characters in CLS were more similar to CPM than PCM,while the rhizome nutrient contents in the process of PES to PCM changed more obviously compared to CPM.As bamboo invasion progressed,the bamboo rhizomes decreased investment in N and P content,which was related to conversion of ATP to proteins,and generated stress reactions including an increase in fine root biomass,length density and so on,meanwhile,adjustment of N,P contents contributed to a rapid adaptation to a heterogeneous environment,realized as fast growth,and showed co-variation in nutrients in the environment (Pan et al.2011;Cai et al.2019).The N,P and C content in native Chinese fir roots decreased as invasion progressed,suggesting they might have adopted a survival strategy of“approach—avoidance conf lict”,such as deep root systems when at a competitive disadvantage (Yao et al.2017).In conclusion,the bamboo rhizome rapidly reacted during the initial contact process with Chinese fir.

Carbon is viewed as playing a skeletal role in plants,and its variation is not significant (An et al.2017).However,this conclusion might not apply to root orders,as in our study,carbon significantly increased in I-Perhizomes,but decreased inClroots during invasion,and the increase in carbon in thePerhizome resulted from the increase in biomass.In the limited underground space,well-developed rhizomes,especially of fine roots,need to invest in more root biomass to occupy more space and thus increase opportunities to obtain more nutrients (Liu et al.2013;Cai et al.2019) and thus outcompete the native species for nutrients.

The architecture of root systems varies with growth,time and function (Yu et al.2010;Xiong et al.2012).However,few studies have focused on the stoichiometric dynamics of root orders during plant invasion.We found that most indices except N:P amongClroot orders in CLS differed significantly or highly significantly,but the traits varied little in mixed interfaces (PCM and CPM).A similar situation occurred amongPerhizome orders;therefore,invasion likely leads to stoichiometric homogeneity within root orders of native and invading species.The mechanism underlying this phenomenon needs to be further explored.In summary,our results ref lected the changing trends in root stoichiometric characters in the species during invasion.

Homeostatic regulation among species and root orders

The mixed interfaces were the main competitive sites for spatial resources for invasive and native species root systems.SpeciesHvalues in forest types are an especially significant measure to reveal the adjustments in strategies for interspecific nutrient competition during invasion.In our study,the homeostasis indices (of both N and P) in mostClandPeroots were greater than 4,indicating that the underground species parts were primarily strongly homeostatic.For dominant species,invasion led to an increase inHN,decrease inHP,and an increase inHN:PofPerhizome orders,but a decrease inHN:PofClroot orders (Fig.4),which might be associated with available nutrients in the mixed interface soil,which had lower AN and higher AP than in the pure forest (Fig.2).Thus,the root systems needed to adjust their nutrient adsorption and turnover to maintain efficient nutrient use (Ulm et al.2017).

Fig.4 Stoichiometric Pe rhizome order trends with bamboo invasion.PES,moso bamboo forest;PCM,moso bamboo forest mixed with few Chinese firs;CPM,Chinese fir forest mixed with few moso bamboos;RO,root order.Letters a (or A) and b (or B) denote significant difference for the same root order at different invasive periods,c (or C) and d (or D) denote significant difference for root orders of each species in the same forest stand.Capital letters denote significant difference at P <0.05,lowercase letters at P <0.01

In the mixed interfaces,I-Pehad higher indexes than did II-PeandCl,which confers a highly competitive advantage for juvenile bamboo in invasion (Jiang et al.2017;Shan et al.2017) and might be a key factor of bamboo invasion success.Jonas et al.(2010) found that plants with high homeostasis would have a conservative strategy for nutrient use,which is more suitable for poor habitat,and conversely,plants with low homeostasis might have an advantage in a changeable habitat.However,this theory might be unsuitable for species with strong physiological integrative ability such as bamboo,and according to our study,the high homeostatic index of I-Peindividuals could be associated with their own high physiological structure adjustment capacity (Li et al.2000).When there is no secondary growth,and the survival cost drops greatly,resulting in a greater competitive advantage.In addition,physiological integration among connected rhizomes of II du bamboo will ensure translocation of nutrients to juveniles (Zhao et al.2 018) and decreased dependence of nutrient absorption from the soil by I-Pe individuals.This phenomenon is particularly prominent in other clonal species (Li et al.2 000;Pan et al.2005;Xu et al.2018),where it has a dominant effect in maintaining nutrient balance in individuals during the growing period.

Root systems have evolved various functional organizations (such as absorptive tissue and transporting tissue)to maximize nutrient absorption and minimize resources required for tissue maintenance and growth (Eissenstat et al.2008).However,nutrient homeostasis in various functional roots is complex.Our results conf irmed that there were some differences in the homeostatic index of root orders in each species (Fig.5).For Chinese fir,root orderHN:Pin pure forest was lower than mixed forests.Correspondingly,bamboo root orderHN:Pin mixed forest was higher than in pure forest.HNandHPin bamboo rhizome orders showed opposite trends,while the native species root order values showed various patterns.Therefore,the physiological response strategies of root systems to invasion were revealed by homeostatic differences.

Invasion effect of bamboo rhizomes on root nutrient limitation in native species

The ratio N:P is a key indicator for determining plant nutrient limiting status and is closely related to soil nutrients(Güsewell 2004).Based on the Koerselman theory,N:P <14,N:P >16 and 14

However,no unified standard of limited threshold values for elemental indicators has yet been put forward (Gong et al.2017).Plant assimilation and adjustment to N and P are more concerned with variation in root allocation (Güsewell 2004).Therefore,the threshold values of limiting nutrients could vary depending on forest landscape,environmental condition and species.For example,Wassen et al.(1995)pointed out that N <14 mg g−1and P <0.7 mg g−1,respectively,were considered to be limiting conditions for plants,whereas Gong et al.(2017) judged a marginal value of about 13 for limiting elements in cotton.Therefore,limiting thresholds for nutrients vary widely among species.In our investigation,there was little positive correlation between soil and plant nutrients,and the dominant species were mainly limited by P before and after bamboo invasion,a result considered to be reliable.However,limiting thresholds of nutrients for various bamboo species still need to be quantified.

Conclusions

Our research showed that bamboo invasion affected the stoichiometric distribution and dynamics of roots and narrowed stoichiometry differences within root orders.During root interactions between invasive and native species,the dominant species revealed high homeostasis.The juvenile bamboo rhizomes had higher homeostatic regulation capacity than adult bamboo and Chinese fir.We consider that a key factor in bamboo invasion success is higher stoichiometric homeostasis in the juveniles.

AcknowledgementsWe are sincerely grateful to Dr Zhao JC of Zhejiang Academy of Forestry and Professor Chen JH of Central South University of Forestry and Technology for technical assistance.We also thank the anonymous reviewers for valuable suggestions to improve the article.