Submitted 18 January 2018Accepted 24 October 2018Published 29 November 2018

Corresponding authorMaiacutera CG Padgurschimairapadgmailcom

Academic editorPaolo Giordani

Additional Information andDeclarations can be found onpage 11

DOI 107717peerj6024

Copyright2018 Padgurschi et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Nitrogen input by bamboos in neotropicalforest a new perspectiveMaiacutera CG Padgurschi1 Simone A Vieira2 Edson JF Stefani1 Gabriela BNardoto3 and Carlos A Joly1

1Plant Biology Department State University of Campinas Campinas Satildeo Paulo Brazil2Center for Environmental Studies and Research State University of Campinas Campinas Satildeo Paulo Brazil3Department of Ecology University of Brasilia Brasilia Brazil

ABSTRACTBackground Nitrogen (N) is an important macronutrient that controls the produc-tivity of ecosystems and biological nitrogen fixation (BNF) is a major source of N interrestrial systems particularly tropical forests Bamboo dominates theses forests butour knowledge regarding the role of bamboo in ecosystem functioning remains in itsinfancy We investigated the importance of a native bamboo species to the N cycle of aNeotropical forestMethods We selected 100 sample units (100 m2 each) in a pristine montane AtlanticForest in BrazilWe counted all the clumps and live culms ofMerostachys neesii bambooand calculated the specific and total leaf area as well as litter production and respectiveN content Potential N input was estimated based on available data on BNF rates forthe same bamboo species whose N input was then contextualized using informationon N cycling components in the study areaResults With 4000 live culms haminus1 the native bamboo may contribute up to 117 kgN haminus1 during summer (January to March) and 196 kg N haminus1 in winter (July toSeptember) When extrapolated for annual values M neesii could contribute morethan 60 kg N haminus1yminus1Discussion The bamboo speciesrsquo contribution to N input may be due to its abundance(habitat availability formicrobial colonization) and the composition of the free-livingNfixer community on its leaves (demonstrated in previous studies) Although some N islost during decomposition this input could mitigate the N deficit in the Atlantic Foreststudied by at least 27 Our findings suggest that M neesii closely regulates N inputand may better explain the high diversity and carbon stocks in the area This is the firsttime that a study has investigated BNF using free-living N fixers on the phyllosphere ofbamboo

Subjects Conservation Biology Ecology Biogeochemistry ForestryKeywords Merostachys neesii Atlantic forest Free-living biological nitrogen fixation N cyclingNeotropical bamboo

INTRODUCTIONWoody bamboos are typical plants in many tropical forests (Humboldt amp Bonpland 1907Judziewicz et al 1999) Their rapid growth under intense levels of light (Cirtain Franklinamp Pezeshki 2009) and leaves with relatively low carbon cost and high photosynthesis rates(Montti et al 2014 Yang et al 2014) result in the widespread occurrence of these plants

How to cite this article Padgurschi MCG Vieira SA Stefani EJF Nardoto GB Joly CA 2018 Nitrogen input by bamboos in neotropicalforest a new perspective PeerJ 6e6024 httpdoiorg107717peerj6024

in forests (Judziewicz et al 1999) Bamboo density effects the dynamics and structureof forests (Tabarelli amp Mantovani 2000 Griscom amp Ashton 2003 Giordano Saacutenchez ampAustin 2009 Rother Rodrigues amp Pizo 2009 Lima et al 2012) serving as a resource fordifferent animals (Reid et al 2004 Areta Bodrati amp Cockle 2009 Hilaacuterio amp Ferrari 2010Cestari amp Bernardi 2011) Although it is unclearwhether they influence ecosystem functionstudies in this regard have increased and demonstrate the role of bamboo in recovering soilfertility (Christanty Kimmins amp Mailly 1997) especially nitrogen (Singh amp Singh 1999Embaye et al 2005 Fukuzawa et al 2006 Watanabe amp Fukuzawa 2013 Shiau et al 2017Borisade amp Odiwe 2018)

Nitrogen (N) controls the productivity and composition of plant species (Townsendet al 2011) and is a limiting factor in many tropical forests (Tanner Vitousek amp Cuevas1998) making N recycling via litter decomposition a key resource in these forests (Vitousekamp Sanford 1986 Kuruvilla Jijeesh amp Seethalakshmi 2014 Borisade amp Odiwe 2018) Therapid growth of bamboo its overabundance and biomass (Yang et al 2014) contributeto nutrient pumping that is nutrients leached into the soil are deposited at the surfaceas bamboo litterfall (Christanty Kimmins amp Mailly 1997) However its intensity dependsprimarily on the ligninN (Tripathi et al 2006) and silicateN ratios of leaves (Watanabe ampFukuzawa 2013) In other words the decomposition rate is greater when N content is highand lignin or silicate levels are low (Tripathi et al 2006Watanabe amp Fukuzawa 2013)

In an agroforestry system in Indonesia the N content in bamboo litterfall varied from282 to 452 kg haminus1 (Mailly Christanty amp Kimmins 1997) with concentrations of 5 to 57 kgN haminus1 yminus1 in other Asian ecosystems (Joshi Sundriyal amp Baluni 1991 Mailly Christantyamp Kimmins 1997) and 332 (Kuruvilla Jijeesh amp Seethalakshmi 2014 Kuruvilla Jijeesh ampSeethalakshmi 2016) to 79 kg N haminus1 in India (Singh amp Singh 1999) Nevertheless thesefigures pale in comparison to the 115 kg N haminus1recorded for Yushania alpina in Ethiopia(Embaye et al 2005) Although there are exceptions (Singh amp Singh 1999 Tripathi et al2006) bamboo litter typically exhibits a high N concentration (Joshi Sundriyal amp Baluni1991 Embaye et al 2005Watanabe amp Fukuzawa 2013 Kuruvilla Jijeesh amp Seethalakshmi2014 Kuruvilla Jijeesh amp Seethalakshmi 2016 Borisade amp Odiwe 2018) but may alsocontain high lignin and silicate levels meaning the N in its litter tends to be releasedgradually over an extended period (Tripathi et al 2006 Watanabe amp Fukuzawa 2013Borisade amp Odiwe 2018)

In addition to N recycling biological nitrogen fixation (BNF) is an important pathwayfor N input in ecosystems (Hedin et al 2009) Studies have shown that free-living BNFfixers in litter and those associated with the aerial parts of plants play a vital role in total Ninputs in tropical forests (Bentley 1987 Benner et al 2007 Reed Cleveland amp Townsend2011) The leaf surfaces (phyllosphere) in these forests harbor a wide range of bacteria(Lambais et al 2006 Fuumlrnkranz et al 2008 Lambais Lucheta amp Crowley 2014) many ofwhich are N fixers and dictate the patterns of N fixation rates (Reed Cleveland amp Townsend2011 Rigonato et al 2016)

In the Brazilian Atlantic Forest (AF) Goacutemez (2012) found a high level of bacterialdiversity in the phyllosphere of Merostachys neesii (Poaceae Bambusoideae) includinggroups of putative free-living diazotrophs that account for a significant amount of

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 218

N fixation Studying the same bamboo species Rigonato et al (2016) reported a highabundance of cyanobacteria from the diazotrophic order Nostacales In this AF area unlikeseveral other studies (Tabarelli amp Mantovani 2000 Griscom amp Ashton 2003 Lima et al2012) the presence of M neesii in a pristine montane forest does not seem to alter theoverall forest structure and diversity (Padgurschi et al 2011) carbon and nitrogen stocks(Vieira et al 2011) or tree biomass (2832 Mg haminus1) (Alves et al 2010) The presence ofMneesii showing evidence of free-living diazotrophs on its leaves suggests that these plantshave efficient mechanisms to cope with potential nutrient limitations in acidic dystrophicsoils (Martins et al 2015)

However disturbances resulting from land use changes may cause an unusualoverabundance of native plants (Pivello et al 2018) including bamboos whichmay also respond positively to CO2 concentration and produce additional biomass(Grombone-Guaratini et al 2013) Moreover human activities such as urbanization andindustrialization produce significant atmospheric N pollution (Souza et al 2015) TheseN additions can have a substantial effect on decomposition rates since they can indirectlyshift the microbial community (Agren Bosatta amp Magill 2001) Thus investigating theinfluence of bamboo on N cycling is key to understanding and predicting ecosystemresponses to global changes

The present paper sought to provide insights on the role of bamboo (M neesii) in thefunctioning of a Neotropical forest The major objectives were (i) to assess the abundanceof bamboo in an Atlantic Forest area (ii) to understand the amount of N added to thesystem by M neesii via free-living diazotrophs in its phyllosphere (iii) to calculate theamount of N that returns to the system through M neesii litter and (iv) to contextualizethe N added by M neesii using information about N cycling components in thestudy area

MATERIALS AND METHODSStudy areaThe study was conducted in an Atlantic Forest region in northeastern Satildeo Paulo stateBrazil in the Serra do Mar State Park (PESM in Portuguese) (Fig 1) We selected 100sample units (100 m2 each) within previously established permanent plots (Joly et al2012) The physiognomy is pristine montane Atlantic Forest (1000 m asl) with a humidsubtropical climate (Cfa and Cfb) average annual temperature of 21 C average annualrainfall of 2180 mm and no dry season (Salemi et al 2013) A dense fog covers the regionalmost daily especially in winter The soil order is Inceptisol (United States Department ofAgriculture taxonomy) with low pH (asymp38) and fertility and high aluminum saturation(Martins et al 2015) Both aboveground biomass (2832 Mg haminus1) (Alves et al 2010) andfloristic diversity (sim200 tree species haminus1) (Padgurschi et al 2011) are high (Joly Metzgeramp Tabarelli 2014) The most abundant families are Arecaceae Myrtaceae Lauraceae andSapotaceae (Padgurschi et al 2011)

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 318

Figure 1 Location of the study area in the context of the Brazilian Atlantic Forest Domain and SerradoMar State Park (45W04prime34primeprime23S17prime24primeprime) Brazilian Atlantic Forest Domain (green) and Serra do MarState Park (red) (PESM in Portuguese) (A) South America with a focus on Brazil In green Atlantic For-est Domain (B) Satildeo Paulo State SE Brazil In red PESM (C) Study area (yellow star) in the context ofPESM

Full-size DOI 107717peerj6024fig-1

Bamboo species density leaf area and litterfallMerostachys neesii Rupr (Poaceae Bambusoideae) a native species of the Brazilian AtlanticForest (Fig 2) prefers humid high-altitude environments (Judziewicz et al 1999) All theclumps and live culms in the 100-sample units were counted (culm density) and culmdensity was compared against the highest density species in the area (Euterpe edulis MartArecaceaemdashPadgurschi et al 2011)

Habitat availability (bamboo leaf area) was estimated in order to determine N inputby free-living diazotrophs in the phyllosphere We calculated the total bamboo leaf area(LAt ) based on (i) culm density (ii) leaf biomass per culm (Lb) and (iii) specific leaf area(SLA) Lb was previously determined by MCG Padgurschi TS Reis LF Alves SA VieiraCA Joly (2018 unpublished data) via destructive harvesting of 20 healthy culms aroundthe study area (Lb= 506 g 95 bootstrap confidence interval 3162 and 7012 were thelowerupper limits respectively) For SLA we randomly chose 50 bamboo leaves in thefield dried at 65 C until constant weight weighed to obtain the dry weight and the leafarea was calculated using an LI-3100 area meter (LI-COR Lincoln Nebraska USA) Theleaf dry weight and leaf area (n= 50) were then used to calculate SLA Leaf area per culm

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 418

Figure 2 Merostachys neesii Rupr (Poaceae Bambusoideae) a native woody bamboo in a pristinemontane forest (Atlantic Forest) Brazil (A) Flowers at anthesis (B) Detail of a clump in the study area(C) Detail of the culm leaf ofM neesii a characteristic of this species Photos MCG Padgurschi

Full-size DOI 107717peerj6024fig-2

(LAc) was determined as follows

LAc = Lb lowastSLA (1)

and total bamboo leaf area (LAt ) (m2 haminus1) by

LAt =LAcn culms

10000(2)

where lsquolsquon culmsrsquorsquo is the culm density within the sample unitsAmong the 100 sample units we randomly selected 40 to install circular litter traps

(022 m2 each) The traps were made of malleable plastic pipes with nylon mesh (2 mm)and supported by PVC pipes about 1 m above the ground The content of the traps was

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 518

collected twice a month over a year from April 2014 to April 2015 sufficient time tocapture this variable (Malhi Doughty amp Galbraith 2011) For each collection the bambooleaves were separated dried (at 65 C until constant weight) and weighed to obtain dryweight We calculated the production of bamboo litterfall in accordance with Sylvestre ampRosa (2002)

LP=

(sumMAlowast10000

CA

)1000

(3)

where LP= annual litter production (kg haminus1yminus1)MA= averagemonthly litter production(kg haminus1) CA= litter collector area (m2) For N chemical analysis of the bamboo leaves werandomly selected three samples for each season (summer fall winter spring) and groundthem to obtain a compound sample per season (results are expressed in kg N haminus1) Theanalysis was performed at the Soil and Plant Laboratory (LAGRO) in Satildeo Paulo Brazilusing the Kjeldahl method of N determination The study was performed with permitsCOTECIF 0103232013 0027662013 and 0106312013 and IBAMASISBIO 33217

Estimating N input by free-living N fixers in the M neesiiphyllosphereTo estimate N input by free-living diazotrophs on bamboo leaves we used BNF ratespreviously recorded in the M neesii phyllosphere at the same site studied here (Goacutemez2012) Goacutemez (2012) estimated BNF rates by acetylene reduction activity (ARA) based ona theoretical conversion ratio of 31 (reduction of three acetylene moles for each N molefixed) (Hardy et al 1968) The BNF rate in the bamboo phyllosphere was 6425 ng N cmminus2

hminus1in winter and 3478 ng N cmminus2 hminus1 in summer and given the significant differencebetween these two values (Goacutemez 2012) calculations for each season were performedseparately

Since light and temperature are important variables that affectmicrobial activity (Bentley1987 Reed Cleveland amp Townsend 2011) we also considered the differences in hours oflight during seasons As such based on available photosynthetically active radiation(PAR) data provided by the Climate and Biosphere LaboratoryDept of AtmosphericSciencesUniversity of Satildeo Paulo bootstrapping (4000 resamplings) was carried out toobtain the median and lowerupper limits of PAR (Table 1) We used the number ofhours around the PAR median added to the lowerupper limits (828 plusmn 70 micromol mminus2 sminus1

in summer 71124 plusmn 55 micromol mminus2 sminus1 in winter 95 confidence intervals) (Table 1)Finally N fixing potential was estimated (Nf expressed in kg N haminus1yminus1) as follows

Nf =(BNF lowastLAt )lowastHl

1012(4)

where Hl is the hours of light in summer or winter (Table 1)

N cyclingTo contextualize the estimated N input mediated byM neesii data on the N cycling in theAtlantic Forest were obtained from the literature The two dominant N input pathways(Hedin et al 2009) considered were symbiotic BNF (Manarin 2012) and total atmospheric

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 618

Table 1 Meteorological data for the study area in 2010 the same year as the BNF rates dataHours of lightday hours with photosynthetically ac-tive radiation (PAR) around the PAR median of the respective seasons months PAR min and PAR max photosynthetically active radiation mini-mum and maximum respectively recorded for that season Median calculated from bootstrapping (4000 resampling) with the 95 confidence in-terval in parentheses

Season Light(hoursday)

PARmin(micromol mminus2 sminus1)

PARmax(micromol mminus2 sminus1)

Median(micromol mminus2 sminus1)

Meantemperature(C)

Accumulatedrainfall(mm)

Summer 9 447 26703 8280 (plusmn70) 193 3804Fall 8 576 22617 7739 (plusmn41) 13 4174Winter 8 378 2064 7112 (plusmn55) 126 2955Spring 9 1392 2640 6026 (plusmn40) 129 692

N deposition (Groppo 2010) in addition to the free-living N fixers on bamboo leaves (thisstudy)

In terms of N required by the system (demand) we used litterfall to predict net primaryproductivity (NPP) The NPP fraction allocated to leaves influences litterfall rates makingit a good predictor of productivity in neotropical forests when the main components ofNPP cannot be measured (Malhi Doughty amp Galbraith 2011) Based on this principle weused the literature data on ecosystem litter production (55 Mg haminus1 yminus1mdashSousa Neto etal 2011) and the N content of the litter (172mdashVieira et al 2011) as well as bamboolitter with its respective nitrogen concentration (see the lsquolsquoBamboo species density leaf areaand litterfallrsquorsquo section for details) The N content of litter is equivalent to the minimumamount required for tree and bamboo growth since plants reallocate nutrients before leafabscission meaning litter exhibits lower N levels when compared to live leaves (ChapinIII et al 1987 Tripathi et al 2006) The annual production of fine roots (lt2 mm) wasconsidered representative of demand These roots represent at least twice as much carbonand nitrogen stock as that found aboveground in the AF (Vieira et al 2011) Fine rootproduction of 10Mg haminus1 yminus1 (Silva 2015) and N content of 13 (Sousa Neto et al 2011)were used

Finally riverine transport and N2O and NO losses via soil emissions were included asoutputs (Groppo 2010 Sousa Neto et al 2011 Ghehi et al 2013) The NO emission wepresented here is based on models developed for a tropical highland forest (Ghehi et al2013) similar to the study area as follows (i) pristine montane forest (1000 m asl) (ii)2000 mm yminus1 of rainfall (iii) presence of bamboo (iv) pH 38 (Ghehi et al 2013Martinset al 2015) All analyses and graph were performed using R environment (R Core Team2014)

RESULTSA total of 579 clumps haminus1 and 4000 live culms haminus1 of M neesii bamboo were countedThe specific leaf area (SLA) was 2044 cm2 gminus1 (95 bootstrap confidence interval19672102 lowerupper limits respectively) which by applying equation one resulted inLAc = 103 m2 and 41 times104 m2 haminus1 of total leaf area (LAt ) for microbial colonizationThese and other data are shown in Table 2

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 718

Table 2 Traits ofM neesii and its contribution to nitrogen input in a pristine montane Atlantic For-est Satildeo Paulo State Brazil Values in parenthesis are lowerupper limits of 95 confidence intervals ob-tained by bootstrapping (1000 resampling)

Merostachys neesii Traits

Density (clumps haminus1) 579Culms (haminus1) 4000(Lw) (g) 011 (010ndash012)LA (cm2) 232 (215ndash252)SLA (cm2 gminus1) 2044 (1967ndash2102)LAc (m2) 103LAt (m2 haminus1) 41times 104

N fixed (kg N haminus1)mdashsummer 117N fixed (kg N haminus1)mdashwinter 196N content in bamboo litterfall () 165

NotesLw Leaf dry weight LA Leaf area SLA Specific leaf area LAc Leaf area per culm (estimated from Eq (1) LAt Total bambooleaf area (estimated from Eq (2) N fixed Total nitrogen fixed on bamboo phyllosphere during summer (Jan Feb Mar) andwinter (Jul Aug Sep) N content in bamboo litterfall of nitrogen in bamboo leaves from litter

Table 3 Estimates of N inputs demand and outputs in the Atlantic Forest studied Except for NO soil emission all the data were obtained fromthe Atlantic Forest area studied

Reference Biome Compartment Nitrogen(kg N haminus1y minus1)

Groppo (2010) Atlantic Forest Brazil Ntotal(N-Ninorg+N-Norg) a 28Manarin (2012) Atlantic Forest Brazil BNF by legume trees 02This study Atlantic Forest Brazil free-living BNF (bamboo leaves) 626

Inputs

Total 656Sousa Neto et al (2011 )Vieira et al 2011

Atlantic Forest Brazil Tree growth 861

This study Atlantic Forest Brazil Bamboo growth 89Sousa Neto et al (2011)Silva (2015)

Atlantic Forest Brazil Fine root (lt2 mm) 1300Demand

Total 2250Groppo (2010) Atlantic Forest Brazil Riverine transport 06Sousa Neto et al (2011) Atlantic Forest Brazil N2O soil emission 08Ghehi et al (2013) Tropical Highland Forest Rwanda NO soil emission 20

Outputs

Total 34Total minus1627

NotesaValue referring to the wet deposition of N in the study area The value presented refers to the average for 2008 and 2009

M neesii can contribute up to 117 kg N haminus1 in summer (January to March) and 196kg N haminus1 in winter (July to September) via free-living diazotrophs on its phyllosphereWhen these values were extrapolated on an annual basis M neesii contributed more than60 kg N haminus1yminus1 representing a decline of at least 278 in the N deficit of the AF westudied (Table 3)

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 818

Figure 3 Seasonal variation ofMneesiirsquos litter production in the pristine montane Atlantic ForestBrazil Significantly higher values are found during summerspring when compared to fallwinter (p lt

0001)Full-size DOI 107717peerj6024fig-3

Annual bamboo litter production was 540 kg haminus1yminus1 with significantly higher valuesin summerspring when compared to fallwinter (plt 0001) (Fig 3) The N content inthis litter fraction was 165 (Table 2) as such the minimum N requirement for bamboogrowth is 89 kg haminus1yminus1 (Table 3)

DISCUSSIONBamboo is important in the recovery of soil physiochemical properties (ChristantyKimmins amp Mailly 1997 Embaye et al 2005 Shiau et al 2017) soil redevelopment(Singh amp Singh 1999) and soil nutrients especially N (Fukuzawa et al 2006 Watanabeamp Fukuzawa 2013 Shiau et al 2017 Borisade amp Odiwe 2018) Its rapid growth andabundance (Yang et al 2014) may contribute to nutrient pumping whereby nutrientsleached deep into the soil are deposited at the surface as bamboo litterfall (ChristantyKimmins amp Mailly 1997)

Although the bamboo density observed here (Table 2) is lower than that found in India(Joshi Sundriyal amp Baluni 1991 Tripathi amp Singh 1994 Christanty Kimmins amp Mailly1997 Singh amp Singh 1999) China (Wang et al 2006) and Ethiopia (Embaye et al 2005) itis similar to that reported in other bamboo forests in the Neotropics (Londontildeo amp Peterson1991Guilherme et al 2004Griscom amp Ashton 2006 Rockwell et al 2014) The abundanceand biomass of M neesii (MCG Padgurschi TS Reis LF Alves SA Vieira CA Joly 2018unpublished data) provide a substantial habitat (leaf area) formicrobial colonization (Table2) which when combined with the composition of the free-living bacterial community onits phyllosphere may influence BNF rates (Benner et al 2007Reed Cleveland amp Townsend2011)

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 918

M neesii exhibits higher cyanobacteria abundance and a larger number of diazotrophsaffiliated to the orderNostocales (Rigonato et al 2016) than E edulis and other species fromthe same area (Goacutemez 2012) Its phyllosphere harbored high annual BNF rates (sim60 kg Nhaminus1 yminus1) almost equal to the rate reported for evergreen tropical forests (Reed Cleveland ampTownsend 2011) but significantly higher than those observed for Spathacanthus hoffmannii(Acanthaceae) Chamaedorea tepejilote (Arecaceae) Brosimum utile (Moraceae) Caryocarcostaricense (Caryocaraceae) Staminodella manilkara (Sapotaceae) Qualea paraensis(Vochysiaceae) and Schizolobium parahybum (Fabaceae) (between 0035 and 5 kg Nhaminus1yminus1mdashFreiberg 1998 Reed Cleveland amp Townsend 2008)

N input by bamboo could mitigate the N deficit in the AF we studied by at least 27(Table 3) where in addition to the low occurrence of tree legumes (Padgurschi et al 2011)the symbiotic BNF rate (02 kg N haminus1 yminus1mdashManarin 2012) is lower than that reported forthe Amazon forest (Nardoto et al 2014) and Costa Rica (Sullivan et al 2014) SymbioticBNF in mature tropical forests may not be as important as previously believed (Sullivanet al 2014 Nardoto et al 2014) making bamboo input particularly relevant since the Ndemand of trees bamboos and fine roots is at least 225 kg N haminus1 yminus1 (Table 3) This is aminimum requirement since only trees with diameter at breast high (DBH) ge 5 cm areincluded with other life forms (such as epiphytes and lianas) excluded from the inventorydata (Joly et al 2012)

Despite the N input of bamboo N demand is high in the system studied here (Table3) and as a result litterfall decomposition plays an important role in nutrition budgeting(Vitousek amp Sanford 1986 Kuruvilla Jijeesh amp Seethalakshmi 2014 Borisade amp Odiwe2018) The annual litter production of M neesii (540 kg haminus1yminus1) is lower than that ofseveral tropical and subtropical bamboo species except for Dendrocalamus strictus (580 kghaminus1mdashJoshi Sundriyal amp Baluni 1991) and Sasa senanensis (600 kg haminus1yminus1mdashWatanabeamp Fukuzawa 2013)

In an agroforestry system in Indonesia the litterfall of different species of the genusGigantochloa ranged from 3 to 47 Mg haminus1 (Mailly Christanty amp Kimmins 1997) in anEthiopian forest the litterfall of Y alpina was 8 Mg haminus1yminus1 (Embaye et al 2005) 12and 19 Mg haminus1 were recorded in Japan for Sasa kurilensis (Tripathi et al 2006) and29 and 69 Mg haminus1 in India (Kuruvilla Jijeesh amp Seethalakshmi 2014 Kuruvilla Jijeeshamp Seethalakshmi 2016) (Singh amp Singh 1999) However since the N content of M neesiilitter (16) was similar to that reported in other studies (12 by Joshi Sundriyal ampBaluni 1991 14 by Embaye et al 2005 14 by Watanabe amp Fukuzawa 2013 15 byKuruvilla Jijeesh amp Seethalakshmi 2014 17 by Kuruvilla Jijeesh amp Seethalakshmi 201617 by Borisade amp Odiwe 2018 07 by Singh amp Singh 1999 09 by Mailly Christantyamp Kimmins 1997 and 1 by Tripathi et al 2006) the final amount of N generated frombamboo litter in each system depends on the annual amount of litter (a total of 89 kg Nhaminus1yminus1 was reported in this study)

Finally it is well known that high N levels and low lignin or silicate concentrationsin leaves increase the decomposition rate of leaf litter (Tripathi et al 2006 Watanabe ampFukuzawa 2013) The leaf lignin content in different bamboo species ranges from 25(Borisade amp Odiwe 2018) tomore than 40 (Tripathi et al 2006Borisade amp Odiwe 2018)

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 1018

with the same observed for silicate (around 20) (Watanabe amp Fukuzawa 2013) As suchit is expected that the N in bamboo litter in the AF is released gradually (Tripathi et al2006 Borisade amp Odiwe 2018) over a period of 3 years or more (Watanabe amp Fukuzawa2013)

CONCLUSIONOur findings suggest that the N fixed by free-living BNF associated with M neesii plays akey role in the functioning of the neotropical forest This may explain the high diversity(Padgurschi et al 2011) carbon and nitrogen stocks (Vieira et al 2011) and biomass(2832 Mg haminus1) (Alves et al 2010) found in the same AF area (Joly Metzger amp Tabarelli2014) contradicting previous studies (Lima et al 2012 Grombone-Guaratini et al 2014)Nonetheless disturbances resulting from human activities such as industrialization andlanduse changesmay increase bamboo abundance (Pivello et al 2018Grombone-Guaratiniet al 2013) Thus the role of bamboo in the overall N cycle in neotropical forests is vitalto understanding ecosystem responses to global change

ACKNOWLEDGEMENTSWe would like to thank Cristina Maguas and Talita Reis for their valuable suggestions andcritical discussion Suzana MS Costa for her help in Fig 1 the students and techniciansengaged in fieldwork the Serra do Mar State Park Santa Virgiacutenia Nucleus for logisticalsupport and to field technician Renato Belinelo for his empirical knowledge of the AtlanticForest which helped us during the field trips We also acknowledge the helpful commentsof two reviewers which have improved this manuscript

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis research was supported by the Brazilian National Research CouncilCNPq (PELD4037102012-0) the British Natural Environment Research CouncilNERC and the SatildeoPaulo Research FoundationFAPESP within the BIOTA Program (201251509-8 and201251872-5) by Coordenacatildeo de Aperfeicoamento de Pessoal de Niacutevel Superior (CAPES)and by Conselho Nacional de Desenvolvimento Cientiacutefico e Tecnoloacutegico (CNPq) via PhDfellowship to Maiacutera CG Padgurschi The meteorological data were provided by theUniversity of Satildeo Paulo with the support of FAPESP projects 201550682-6 201251872-5201250343-9 200850285-3 200757465-4 2003 12595-7 There was no additionalexternal funding received for this study The funders had no role in study design datacollection and analysis decision to publish or preparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsBrazilian National Research CouncilCNPq PELD 4037102012-0

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 1118

British Natural Environment Research CouncilNERC and Satildeo Paulo ResearchFoundationFAPESP 201251509-8 201251872-5Coordenacatildeo de Aperfeicoamento de Pessoal de Niacutevel Superior (CAPES)Conselho Nacional de Desenvolvimento Cientiacutefico e Tecnoloacutegico (CNPq)Satildeo Paulo Research FoundationFAPESP 201550682-6 201251872-5 201250343-9200850285-3 200757465-4 2003 12595-7

Competing InterestsSimone A Vieira and Gabriela B Nardoto are Academic Editors for PeerJ

Author Contributionsbull Maiacutera CG Padgurschi conceived and designed the experiments performed theexperiments analyzed the data contributed reagentsmaterialsanalysis tools preparedfigures andor tables authored or reviewed drafts of the paper approved the final draftbull Simone A Vieira conceived and designed the experiments analyzed the data contributedreagentsmaterialsanalysis tools authored or reviewed drafts of the paper approved thefinal draftbull Edson JF Stefani conceived and designed the experiments performed the experimentsauthored or reviewed drafts of the paper approved the final draftbull Gabriela B Nardoto authored or reviewed drafts of the paper approved the final draftreviewed it critically for important intellectual contentbull Carlos A Joly authored or reviewed drafts of the paper approved the final draft

Field Study PermissionsThe following information was supplied relating to field study approvals (ie approvingbody and any reference numbers)

The research was performed with permits COTECIF 0103232013 0027662013 and0106312013 and IBAMASISBIO 33217

Data AvailabilityThe following information was supplied regarding data availability

The raw data are provided in a Supplemental File

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj6024supplemental-information

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stand effects of inorganic nitrogen on litter decomposition Oecologia 12894ndash98DOI 101007s004420100646

Alves LF Vieira SA Scaranello MA Camargo PB Santos FAM Joly CA Martinelli LA2010 Forest structure and live aboveground biomass variation along an elevational

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gradient of tropical Atlantic moist forest (Brazil) Forest Ecology and Management260(5)679ndash691 DOI 101016jforeco201005023

Areta JI Bodrati A Cockle K 2009 Specialization on Guadua bamboo seeds bythree bird species in the Atlantic Forest of Argentina Biotropica 41(1)66ndash73DOI 101111j1744-7429200800458x

Benner JW Conroy S Lunch CK Toyoda N Vitousek PM 2007 Phosphorus fer-tilization increases the abundance and nitrogenase activity of the cyanolichenPseudocyphellaria crocata in Hawaiian montane forests Biotropica 39(3)400ndash405DOI 101111j1744-7429200700267x

Bentley BL 1987 Nitrogen fixation by epiphylls in a tropical rainforest Annals of theMissouri Botanical Garden 74(2)234ndash241 DOI 1023072399396

Borisade T Odiwe A 2018 Nutrient input in litters and soil of bambusa vulgarisstands in asecondary rainforest ile-ife Nigeria Journal of Tropical Forest Science30(2)195ndash206 DOI 1026525jtfs2018302195206

Cestari C Bernardi CJ 2011 Predation of the buffy-fronted seedeater Sporophilafrontalis (Aves Emberizidae) onMerostachys neesii (Poaceae Babusoideae) seedsduring a masting event in the Atlantic forest Biota Neotropica 11(3)407ndash411DOI 101590S1676-06032011000300033

Chapin III FS Bloom AJ Field CBWaring RH 1987 Plant responses to multipleenviromental factors BioScience 37(1)49ndash57 DOI 1023071310177

Christanty L Kimmins JP Mailly D 1997 lsquolsquoWithout bamboo the land diesrsquorsquo a concep-tual model of the biogeochemical role of bamboo in an Indonesian agroforestry sys-tem Forest Ecology and Management 9183ndash91 DOI 101016S0378-1127(96)03881-9

CirtainMC Franklin SB Pezeshki SR 2009 Effect of light intensity on Arundinariagigantea growth and physiology Castanea 74(3)236ndash246 DOI 10217908-060R31

Embaye KWeihM Ledin S Christersson L 2005 Biomass and nutrient distributionin a highland bamboo forest in southwest Ethiopia implications for managementForest Ecology and Management 204159ndash169 DOI 101016jforeco200407074

Freiberg E 1998Microclimatic parameters influencing nitrogen fixation in thephyllosphere in a Costa Rican premontane rain forest Oecologia 117(1ndash2)9ndash18DOI 101007s004420050625

Fukuzawa K Shibata H Takagi K NomuraM Kurima N Fukazawa T Satoh F Sasa K2006 Effect of clear-cutting on nitrogen leaching and fine root dynamics in a cool-temperate forested watershed in northern Japan Forest Ecology and Management225257ndash261 DOI 101016jforeco200601001

FuumlrnkranzMWanekW Richter A Abell G Rasche F Sessitsch A 2008 Nitrogenfixation by phyllosphere bacteria associated with higher plants and their colonizingepiphytes of a tropical lowland rainforest of Costa Rica ISME Journal 2(5)561ndash570DOI 101038ismej200814

Ghehi NGWerner C Hufkens K Kiese R Ranst E Nsabimana DWallin G Klemedts-son L Butterbach-Bahl K Boeckx P 2013 Detailed regional predictions of N2Oand NO emissions from a tropical highland rainforest Biogeosciences Discussions101483ndash1516

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 1318

Giordano CV Saacutenchez RA Austin AT 2009 Gregarious bamboo flowering opens awindow of opportunity for regeneration in a temperate forest of Patagonia NewPhytologist 181880ndash889 DOI 101111j1469-8137200802708x

Goacutemez SPM 2012 Diversidade de bacteacuterias diazotroacuteficas e fixacatildeo bioloacutegica donitrogecircnio na Mata Atlacircntica D Phil thesis University of Satildeo Paulo

Griscom BW Ashton PMS 2003 Bamboo control of forest succession Guaduasarcocarpa in Southeastern Peru Forest Ecology and Management 175(1ndash3)445ndash454DOI 101016S0378-1127(02)00214-1

Griscom BW Ashton PMS 2006 A self-perpetuating bamboo disturbance cycle in aneotropical forest Journal of Tropical Ecology 22(05)587ndash597DOI 101017S0266467406003361

Grombone-Guaratini MT Alves LF Vinha D Antocircnio G Correcirca D 2014 Seed rain inareas with and without bamboo dominance within an urban fragment of the AtlanticForest Acta Botanica Brasilica 28(1)76ndash85 DOI 101590S0102-33062014000100008

Grombone-Guaratini MT Gaspar M Oliveira VF Torres MAMG Nascimento AAidar MPM 2013 Atmospheric CO2 enrichment markedly increases photosynthesisand growth in a woody tropical bamboo from the Brazilian Atlantic Forest NewZealand Journal of Botany 51(4)275ndash285 DOI 1010800028825X2013829502

Groppo JD 2010 Caracterizacatildeo hidroloacutegica e dinacircmica do nitrogecircnio em uma microba-cia com cobertura florestal (Mata Atlacircntica) no Parque Estadual da Serra do Marnuacutecleo Santa Virgiacutenia D Phil Thesis University of Satildeo Paulo

Guilherme FAG Oliveira-Filho AT Appolinaacuterio V Bearzoti E 2004 Effects offlooding regime and woody bamboos on tree community dynamics in a sectionof tropical semideciduous forest in South-Eastern Brazil Plant Ecology 17419ndash36DOI 101023BVEGE000004605197752cd

Hardy RWF Holsten RD Jackson EK Burns RC 1968 The acetylenendashethylene assayfor N2 fixation laboratory and field evaluation Plant Physiology 431185ndash1207DOI 101104pp4381185

Hedin LO Brookshire ENJ Menge DNL Barron AR 2009 The nitrogen paradox inTropical Forest ecosystems Annual Review of Ecology Evolution and Systematics40(1)613ndash635 DOI 101146annurevecolsys37091305110246

Hilaacuterio RR Ferrari SF 2010 Feeding ecology of a group of buffy-headed marmosets(Callithrix flaviceps) fungi as a preferred resource American Journal of Primatology72(6)515ndash521

Humboldt A Bonpland A 1907 Personal narrative of travels to the equinoctial regions ofAmerica during the years 1799ndash1804 2nd edition London George Bell amp Sons

Joly CA Assis MA Bernacci LA Tamashiro JY De CamposMCR Gomes JAMALacerdaMS Santos FAM Pedroni F Pereira LS Padgurschi MDCG Prata EMBRamos E Torres RB Rochelle AC Martins FR Alves LF Vieira SA MartinelliLA Camargo PB Aidar MPM Eisenlohr PV Simotildees E Villani JP Belinello R2012 Floristic and phytosociology in permanent plots of the Atlantic rainforestalong an altitudinal gradient in southeastern Brazil Biota Neotropica 12(1)125ndash145DOI 101590S1676-06032012000100012

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 1418

Joly CA Metzger JP Tabarelli M 2014 Experiences from the Brazilian Atlantic Forestecological findings and conservation initiatives New Phytologist 204459ndash473DOI 101111nph12989

Joshi AP Sundriyal RC Baluni DC 1991 Nutrient dynamics of a lower Siwalikbamboo forest in the Garhwal Himalaya India Journal of Tropical Forest Science3(3)238ndash250

Judziewicz EJ Clark LG Londontildeo X SternMJ 1999 American bamboos WashingtonSmithsonian Institution Press

Kuruvilla T Jijeesh CM Seethalakshmi KK 2014 Litter production decompositionand nutrient mineralization dynamics of Ochlandra setigera a rare bamboo speciesof Nilgiri Biosphere Reserve India Journal of Forestry Research 25(3)579ndash584DOI 101007s11676-014-0497-3

Kuruvilla T Jijeesh CM Seethalakshmi KK 2016 Litter production and decompositiondynamics of a rare and endemic bamboo speciesMunrochloa ritcheyi of WesternGhats India Tropical Ecology 57(3)601ndash606

Lambais MR Crowley DE Cury JC Buumlll RC Rodrigues RR 2006 Bacterial di-versity in tree canopies of the Atlantic Forest Science 312(5782)1917ndash1917DOI 101126science1124696

Lambais MR Lucheta AR Crowley DE 2014 Bacterial community assemblagesassociated with the phyllosphere dermosphere and rhizosphere of tree speciesof the Atlantic forest are host taxon dependentMicrobial Ecology 68(3)567ndash574DOI 101007s00248-014-0433-2

Lima RAF Rother DC Muler AE Lepsch IF Rodrigues RR 2012 Bamboo overabun-dance alters forest structure and dynamics in the Atlantic forest hotspot BiologicalConservation 147(1)32ndash39 DOI 101016jbiocon201201015

Londontildeo X Peterson PM 1991 Guadua sarcocarpa (Poaceae Bambuseae) a newspecies of Amazonian bamboo with fleshy fruits Systematic Botany 16(4)630ndash638DOI 1023072418866

Mailly D Christanty L Kimmins JP 1997 lsquolsquoWithout bamboo the land diesrsquorsquo nutrientcycling and biogeochemistry of a Javanese bamboo talun-kebun system ForestEcology and Management 91155ndash173 DOI 101016S0378-1127(96)03893-5

Malhi Y Doughty C Galbraith D 2011 The allocation of ecosystem net primaryproductivity in tropical forests Philosophical transactions of the Royal Society ofLondon Series B 3663225ndash3245 DOI 101098rstb20110062

Manarin EC 2012 Potencial de fixacatildeo de nitrogecircnio por leguminosas noduladas ecianobacteacuterias terrestres na Mata Atlacircntica SP MSc Thesis University of Campinas

Martins SC Sousa Neto E Piccolo MC Almeida DQA Camargo PB Carmo JDBPorder S Lins SRMMartinelli LA 2015 Soil texture and chemical characteristicsalong an elevation range in the coastal Atlantic forest of Southeast Brazil GeodermaRegional 5106ndash116 DOI 101016jgeodrs201504005

Montti L Villagra M Campanello PI Gatti MG Goldstein G 2014 Functional traitsenhance invasiveness of bamboos over co-occurring tree saplings in the semidecidu-ous Atlantic Forest Acta Oecologica 5436ndash44 DOI 101016jactao201303004

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 1518

Nardoto GB Quesada CA Patintildeo S Saiz G Baker TR Schwarz M Schrodt FFeldpausch TR Domingues TF Marimon BS Junior BM Vieira ICG Sil-veira M BirdMI Phillips OL Lloyd J Martinelli LA 2014 Basin-wide vari-ations in Amazon forest nitrogen-cycling characteristics as inferred from plantand soil 15N14Nmeasurements Plant Ecology and Diversity 7(1ndash2)173ndash187DOI 101080175508742013807524

Padgurschi MDCG Pereira LDS Tamashiro JY Joly CA 2011 Floristic compositionand similarity between areas of Montane Atlantic rainforest Satildeo Paulo Brazil BiotaNeotropica 11(2)139ndash152 DOI 101590S1676-06032011000200014

Pivello VR Vieira MV Grombone-Guaratini MT Matos DMS 2018 Thinking aboutsuper-dominant populations of native speciesmdashexamples from Brazil Perspectives inEcology and Conservation 16(2)74ndash82 DOI 101016jpecon201804001

R Core Team 2014 R a language and environment for statistical computing Vienna RFoundation for Statistical Computing Available at httpwwwR-projectorg

Reed SC Cleveland CC Townsend AR 2008 Tree species control rates of free-living nitrogen fixation in a tropical rain forest Ecology 89(10)2924ndash2934DOI 10189007-14301

Reed SC Cleveland CC Townsend AR 2011 Functional ecology of Free-living nitrogenfixation a contemporary perspective Annual Review of Ecology Evolution andSystematics 42(1)489ndash512 DOI 101146annurev-ecolsys-102710-145034

Reid S Diaz IA Arnesto JJ WilsonMF 2004 Importance of native bamboo forunderstory birds in chilean temperate forests The Auk Ornithological Advances121(2)515ndash525 DOI 1016420004-8038(2004)121[0515IONBFU]20CO2

Rigonato J Goncalves N Andreote APD Lambais MR Fiore MF 2016 Esti-mating genetic structure and diversity of cyanobacterial communities in At-lantic forest phyllosphere Canadian Journal of Microbiology 62(11)953ndash960DOI 101139cjm-2016-0229

Rockwell CA Kainer KA DrsquoOliveira MVN Staudhammer CL Baraloto C 2014Logging in bamboo-dominated forests in southwestern Amazonia caveats andopportunities for smallholder forest management Forest Ecology and Management315202ndash210 DOI 101016jforeco201312022

Rother DC Rodrigues RR PizoMA 2009 Effects of bamboo stands on seed rain andseed limitation in a rainforest Forest Ecology and Management 257(3)885ndash892DOI 101016jforeco200810022

Salemi LP Groppo JD Trevisan R Moraes JM Ferraz SFB Villani JP Duarte-NetoPJ Martinelli LA 2013 Land-use change in the Atlantic rainforest region conse-quences for the hydrology of small catchments Journal of Hydrology 499100ndash109DOI 101016jjhydrol201306049

Shiau YWang H Chen T Jien S Tian G Chiu C 2017 Improvement in the biochem-ical and chemical properties of badland soils by thorny bamboo Scientific Reports740561 DOI 101038srep40561

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 1618

Silva CA 2015 Estoque e producatildeo de raiz fina ao longo de um gradiente altitudinalde floresta atlacircntica na serra do mar Satildeo Paulo Brasil MSc Thesis University ofCampinas

Singh AN Singh JS 1999 Biomass net primary production and impact of bambooplantation on soil redevelopment in a dry tropical region Forest Ecology andManagement 119195ndash207 DOI 101016S0378-1127(98)00523-4

Sousa Neto E Carmo JB Martins SC Alves LF Vieira SA Piccolo MC Camargo PBCouto HTZ Joly CA Martinelli LA 2011 Soil-atmosphere exchange of nitrousoxide methane and carbon dioxide in a gradient of elevation in the coastal BrazilianAtlantic Forest Biogeosciences 8(3)733ndash742 DOI 105194bg-8-733-2011

Souza PA Ponette-Gonzaacutelez AG MelloWZWeathers KC Santos IA 2015 At-mospheric organic and inorganic nitrogen inputs to coastal urban and montaneAtlantic Forest sites in southeastern Brazil Atmospheric Research 160126ndash137DOI 101016jatmosres201503011

Sullivan BW SmithWK Townsend AR NastoMK Reed SC Chazdon RL Cleve-land CC 2014 Spatially robust estimates of biological nitrogen (N) fixationimply substantial human alteration of the tropical N cycle Proceedings of theNational Academy of Sciences of the United States of America 111(22)8101ndash8106DOI 101073pnas1320646111

Sylvestre LDS RosaMD 2002Manual metodoloacutegico para estudos botacircnicos na MataAtlacircntica Seropeacutedica EDUR

Tabarelli M MantovaniW 2000 Gap-phase regeneration in a tropical montane Forestthe effects of gap structure and bamboo species Plant Ecology 148(2)149ndash155DOI 101023A1009823510688

Tanner EVJ Vitousek PM Cuevas E 1998 Experimental investigation of nutrientlimitation of forest growth on wet tropical mountains Ecology 79(1)10ndash22DOI 1018900012-9658(1998)079[0010EIONLO]20CO2

Townsend AR Cleveland CC Houlton BZ Alden CBWhite JW 2011Multi-elementregulation of the tropical forest carbon cycle Frontiers in Ecology and the Environ-ment 9(1)9ndash17 DOI 101890100047

Tripathi SK Singh KP 1994 Productivity and nutrient cycling in recently harvested andmature bamboo savannas in the dry tropics Journal of Applied Ecology 31109ndash124DOI 1023072404604

Tripathi SK Sumida A Shibata H Ono K Uemura S Kodama Y Hara T 2006Leaf litterfall and decomposition of different above-belowground parts of birch(Betula ermanii ) trees and dwarf bamboo (Sasa kurilensis) shrubs in a youngsecondary forest in Northern Japan Biology and Fertility of Soils 43237ndash246DOI 101007s00374-006-0100-y

Vieira SA Alves LF Duarte-Neto PJ Martins SC Veiga LG Scaranello MAS PicolloMC Camargo PB Carmo JB Sousa Neto E Santos FAM Joly CA MartinelliLA 2011 Stocks of carbon and nitrogen and partitioning between above- andbelowground pools in the Brazilian coastal Atlantic Forest elevation range Ecologyand Evolution 1(3)421ndash434 DOI 101002ece341

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 1718

Vitousek PM Sanford RL 1986 Nutrient cycling in moist Tropical Forest AnnualReview of Ecology and Systematics 17(1)137ndash167DOI 101146annureves17110186001033

WangW Franklin SB Ren Y Ouellette JR 2006 Growth of bamboo Fargesiaqinlingensis and regeneration of trees in a mixed hardwood-conifer forest inthe Qinling Mountains China Forest Ecology and Management 234107ndash115DOI 101016jforeco200606028

Watanabe T Fukuzawa K 2013 Temporal changes in litterfall litter decom-position and their chemical composition in Sasa dwarf bamboo in a naturalforest ecosystem of northern Japan Journal of Forest Research 18129ndash138DOI 101007s10310-011-0330-1

Yang S SunM Zhang Y Cochard H Cao K 2014 Strong leaf morphologicalanatomical and physiological responses of a subtropical woody bamboo(Sinarundinaria nitida) to contrasting light environments Plant Ecology215(1)97ndash109 DOI 101007s11258-013-0281-z

Padgurschi et al (2018) PeerJ DOI 107717peerj6024 1818