Article

Divergent Adaptation Strategies of Vascular FacultativeEpiphytes to Bark and Soil Habitats Insightsfrom Stoichiometry

Tingting Zhang 12 Wenyao Liu 13 Tao Hu 12 Dandan Tang 4 Yuxuan Mo 12 and Yi Wu 13

Citation Zhang T Liu W Hu T

Tang D Mo Y Wu Y Divergent

Adaptation Strategies of Vascular

Facultative Epiphytes to Bark and

Soil Habitats Insights from

Stoichiometry Forests 2021 12 16

httpsdxdoiorg103390f1201

0016

Received 1 December 2020

Accepted 23 December 2020

Published 24 December 2020

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1 CAS Key Laboratory of Tropical Forest Ecology Xishuangbanna Tropical Botanical Garden Chinese Academyof Sciences Kunming 650223 China zhangtingtingxtbgaccn (TZ) hutaoxtbgaccn (TH)moyuxuanxtbgaccn (YM) wuyixtbgaccn (YW)

2 University of Chinese Academy of Sciences Beijing 100049 China3 Center of Plant Ecology Core Botanical Gardens Chinese Academy of Sciences

Xishuangbanna 666303 China4 Qianxinan State Nanpanjiang Forest Farm Xingyi 562400 China tangdandanxtbgaccn Correspondence liuwyxtbgaccn

Abstract Understanding the stoichiometric traits of plants is critical for studying their ecologicaladaptation strategies Facultative epiphytes (which can also live on the ground) are an importantcomponent of epiphytic flora of montane forest ecosystems However a key gap persists in ourunderstanding how facultative epiphytes can adapt different nutritional conditions of ground andcanopy habitats To study adaptive strategies of facultative epiphytes and the characteristics of thecontent and stoichiometric homeostasis of C N and P elements we conducted a field experimentand a greenhouse N and P additions cultivation experiment We found that epiphytic individuals offacultative epiphytes showed lower CN and CP ratios higher variation in elemental compositionand more pronounced N limitation than terrestrial individuals Moreover facultative epiphytesshowed strong control over the elemental composition of leaves and their stoichiometric homeostasisof leaves and stems were stronger than roots Furthermore the homeostasis of facultative epiphytesdecreased in the order N gt P Our results indicated that epiphytic and terrestrial individuals offacultative epiphytes have difference in nutrient limitation and they use plastic strategies in differenthabitats Epiphytic individuals survive in the intermittent habitat through luxury consumption ofnutrient while terrestrial individuals were relatively conservative nutrient users Furthermore ourresults implied that facultative epiphytes maintain stable metabolic leaf activity via variable elementconcentrations of roots to adapt to highly heterogeneous forest habitats

Keywords facultative epiphytes nutrient use strategy ecological stoichiometry stoichiometrichomeostasis carbon nitrogen phosphorus

1 Introduction

As an essential component of the forest canopy epiphytes are important to the floristicdiversity structure and function of moist mountainous forest ecosystems and play avital role in maintaining ecosystem biodiversity and the circulation of nutrients [1ndash3]Facultative epiphytes are an important life history group being special epiphytes thatcan grow both on host trees and ground [4ndash6] In terrestrial habitats the primary role ofinterspecific interaction was competition while species in harsh epiphytic habitats dependon each other to survive [7] Furthermore epiphytic habitats are often characterized bylimited water and nutrient storage capacity sporadic nutrient inputs low physical stabilityvariable temperature and humidity [8ndash10] while terrestrial habitats are often characterizedby relatively constant nutrient and water supply [51112] Therefore facultative epiphytesmust not only adapt to a harsh canopy environment but also be well adapted to therelatively stable environment under the forest Recent work shows that the morphological

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Forests 2021 12 16 2 of 15

anatomical physiological and growth traits between terrestrial and epiphytic individualsof facultative epiphytes have undergone significant plasticity changes in adapting to canopyand terrestrial habitats [513] However whether there are differences in stoichiometriccharacteristics and nutrient use strategies between epiphytic and terrestrial epiphytes offacultative epiphytes remains little known

Epiphytes are generally considered to survive in the harsh habitats through luxuryconsumption of nutrients [14] Therefore epiphytes may show higher N and P concen-trations and lower nutrient use than terrestrial plants [1214] However little researchcompared the difference in nutrient use between epiphytes and terrestrial plants and manyof them investigated different species and result in a potential phylogenetic bias [12] Fac-ultative epiphytes can live both in epiphytic and terrestrial habitats and effectively usingboth canopy and ground resources to maintain their competitiveness in both habitats [15]Therefore facultative epiphytes make an excellent system for the better understanding ofthe adaptation and nutrient trade-offs of plants in the epiphytic and terrestrial habitats [12]

Ecological stoichiometry mainly studies the balance of energy and multiple chemicalelements in biological systems [1617] It is a fast-emerging tool that is critical for studyingthe ability of plants to maintain constant elemental composition [18ndash21] the ecologicalstrategies of plants [22] and their nutrient limitations [23ndash25] in natural and semi-naturalecosystems The conservative species generally showed low N and P content and high CNand CP ratios while luxury species showed the opposite traits [142627] Moreover theNP of plants has long been considered to be an indicator of nutrient limitation Koerselmanand Meuleman [28] proposed that NP lt 14 indicates plants are N-limited and NP gt 16indicates they are P-limited Wanek and Zotz [29] argued that NP gt 12 indicates P limitationor co-limitation of N and P in epiphytes NP lt 12 indicates N limitation

Stoichiometric homeostasis (H) refers to the ability of an organism to maintain rela-tively constant elemental concentrations and ratios despite changes in its habitat [1730]The ecological stoichiometric homeostasis of plants mainly affected by plant species or-gans or tissues and element types [1722] Stoichiometric homeostasis not only reflectsthe stability of plants but it also may be used to analyze their environmental adaptationstrategies [21] Plants with a stronger stoichiometric homeostasis are more conservative intheir nutrient use than those plants with weaker stability [2231] However the latter aremore adaptable to variable environments while the former may be better suited to a stableenvironment [3233] The facultative epiphytes may therefore show weak stoichiometrichomeostasis to adapt to variable habitats Furthermore an element with high content hashigher H than an element with low content for example HN is higher than HP [34ndash36]

The aboveground parts of vascular plants have stronger stoichiometric homeostasisthat is negatively correlated with that of their belowground parts reflecting the ability ofplants to adapt to their environments by adjusting the nutrient distribution of different or-gans [2237] However since most research has mainly focused on leaves far less is knownabout the stoichiometric homeostasis of stems and roots [2038] which is important forfully understanding the ecological strategies of plant species [22] Different organs exhibitdifferent stoichiometric characteristics because of their functional differentiation [16] Interrestrial plants (eg tree seedlings and Arabidopsis thaliana (L) Heynh) as the chief pho-tosynthetic organs leaves exert stronger control over their elemental composition than doeither the stems or roots whose primary role is nutrient acquisition and storage [162038]Yet whether this pattern also generally applies to epiphytes needs further study

The C N and P stoichiometric patterns of terrestrial plants have been extensivelystudied at regional [39] national [40] and global scales [41] However the ecologicalstoichiometry traits of epiphytes dwelling in the forest canopy remain little known de-spite the vital role these plants play in the nutrient and energy cycling of subtropicalforest ecosystems [42] To date only a few studies focused the stoichiometric traits ofepiphytes [2943] no studies have reported the stoichiometric homeostasis of facultativeepiphytes Moreover according to Zotz [12] epiphytes are luxury consumption plants thathave high nutrient concentrations but low rates of photosynthesis which general feature

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a low nutrient use efficiency [14] However whether epiphytic individuals of facultativeepiphytes (which occur both epiphytically and terrestrially) are more efficient in nutrientuse than terrestrial individuals remain an open question In this research to study theecological stoichiometric characteristics and homeostasis of C N and P in three commonvascular facultative epiphytes in the montane moist evergreen broad-leaved forest of AilaoMountain National Nature Reserve we conducted a field experiment and a greenhousecultivation experiment We proposed the following hypotheses (1) There are significantdifferences in adaptation strategy between the epiphytic and terrestrial individuals offacultative epiphytes which reflect in their stoichiometry pattern (2) Facultative epiphytesexhibit weak stoichiometric homeostasis to adapt to variable habitats (3) and they showstronger control on the elemental composition of leaves than that of roots

2 Materials and Methods21 Study Area and Species

This study was conducted in subtropical montane moist evergreen broad-leaved forestin Xujiaba the core area of the Ailao Mountain National Natural Reserve Southwest China(2336primendash2456prime N 10044primendash10130prime E) The Ailao Mountain have the largest most intactmountain moist evergreen broad-leaved forest in China forming an important portion ofthis countryrsquos subtropical forest ecosystem [44] In this montane moist evergreen broad-leaved forest the canopy and trunk are rich in epiphytic plant communities [43] providinga natural and ideal place to study the stoichiometric characteristics of epiphytes The AilaoMountain are located in a climate transition area in the central and northern parts ofYunnanrsquos subtropical zone characterized by dry and rainy seasons due to the influence ofthe southwest monsoon According to long-term monitoring data from the Ailao MountainEcological Station of the Chinese Academy of Sciences the annual precipitation in this areais 1947 mm with an annual evaporation of 1192 mm and the dry season (NovemberndashApril)and rainy season (MayndashOctober) are distinct The precipitation during the rainy seasonaccounts for ca 85 of the annual precipitation at the site where the relative humidity is85 annual average temperature is 113 C monthly average maximum temperature is157 C and monthly average minimum temperature is 56 C [45]

Xujiaba area is rich in epiphytes there are nearly 600 species including lichens (183)bryophytes (176) ferns (117) and spermatophytes (113) [843] The dominant facultative epi-phytic spermatophytes are Cautleya gracilis (Smith) Dandy (an annual erect herb 025ndash08 min height) Elatostema monandrum (D Don) Hara (a small annual herb 005ndash02 m in height)and Briggsia longifolia var multiflora (a perennial herb without stem) [43] In this areaaccording to our survey epiphytic individuals of facultative epiphytes are usually foundon tree trunks with moss mat or thicker humus soil while terrestrial individuals are usuallydistributed in the forest floor under canopy gap (Figure 1) Most individuals of C gracilisgrow on the forest floor while E monandrum and B longifolia show the inverse pattern Thedistribution of facultative epiphytes may be affected by the seed supply and microclimaticconditions [46]

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Figure 1 The pictures of investigated species (AB) epiphytic and terrestrial individuals of Cautleya gracilis (CD) epiphytic and terrestrial individuals of Elatostema monandrum (EF) epi-phytic and terrestrial individuals of Briggsia longifolia respectively

22 Field Sampling and Greenhouse Culture In the field sampling experiment we used the natural nutrient gradients of the forest

canopy and ground soil stands to carry out nutrient additions Many factors such as light substrate water and nutrient content jointly affect the content of chemical elements in plants in the field [3234] so the relationship between a plantrsquos elemental content and its growth substrate cannot be accurately estimated by field sampling alone Therefore to accurately evaluate the stoichiometric stability of facultative epiphytes we also per-formed a greenhouse cultivation experiment in addition to the field experiment

Field sampling was done during the rainy season (August 2018) Four collection line transects were set along four evenly divided directions in the core area of montane moist evergreen broad-leaved forest in the Ailao Mountains and 3ndash5 sampling points were selected per line transect We defined individuals that grow on the trunk as epiphytic individuals and individuals that grow on the ground as terrestrial individuals The healthy mature terrestrial and epiphytic individuals of three dominant facultative epi-phytes (C gracilis E monandrum and B longifolia) were separately collected from each sampling point for which terrestrial and epiphytic individuals were collected from the forests soil and c 15 m of their host trees respectively In total 34 samples (16 samples from epiphytic individuals and 18 samples from terrestrial individuals) were collected

Figure 1 The pictures of investigated species (AB) epiphytic and terrestrial individuals of Cautleya gracilis (CD)epiphytic and terrestrial individuals of Elatostema monandrum (EF) epiphytic and terrestrial individuals of Briggsia longifoliarespectively

22 Field Sampling and Greenhouse Culture

In the field sampling experiment we used the natural nutrient gradients of the forestcanopy and ground soil stands to carry out nutrient additions Many factors such as lightsubstrate water and nutrient content jointly affect the content of chemical elements inplants in the field [3234] so the relationship between a plantrsquos elemental content and itsgrowth substrate cannot be accurately estimated by field sampling alone Therefore toaccurately evaluate the stoichiometric stability of facultative epiphytes we also performeda greenhouse cultivation experiment in addition to the field experiment

Field sampling was done during the rainy season (August 2018) Four collectionline transects were set along four evenly divided directions in the core area of montanemoist evergreen broad-leaved forest in the Ailao Mountains and 3ndash5 sampling points wereselected per line transect We defined individuals that grow on the trunk as epiphyticindividuals and individuals that grow on the ground as terrestrial individuals Thehealthy mature terrestrial and epiphytic individuals of three dominant facultative epiphytes(C gracilis E monandrum and B longifolia) were separately collected from each sampling

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point for which terrestrial and epiphytic individuals were collected from the forests soiland c 15 m of their host trees respectively In total 34 samples (16 samples from epiphyticindividuals and 18 samples from terrestrial individuals) were collected for C gracilis 30samples (18 samples from epiphytic individuals and 12 samples from terrestrial individuals)for E monandrum and 38 samples (20 samples from epiphytic individuals and 18 samplesfrom terrestrial individuals) for B longifolia We collected whole plant individuals whoseroots stems and leaves were then separated and stored as well the soil at their growthpositions of the target species for use a growing substrate for these plants These plantsamples were first washed with tap water then washed with distilled water and placed intoenvelopes and oven-dried to a constant weight (48 h) at 65 C then the dried samples wereground to powder The soil samples were dried ground and passed through a 60-meshsieve before their analysis

The cultivation experiment was carried out in a greenhouse from April to August2018 In this experiment the N and P fertilizers used were respectively NH4NO3 andNaH2PO4 their fertilization gradients are shown in Table 1 which consisted of ninetreatments in total with three replicates per treatment There were five N levels (05 1 24 and 6 mmol Lminus1) under a moderate P gradient (025 mmol Lminus1) and five P gradients(00625 0125 025 05 and 075 mmol Lminus1) under a moderate N gradient (2 mmol Lminus1) Itis difficult to collect and cultivate seedlings of C gracilis thus only the other two specieswere used for the cultivation experiment Healthy epiphytic seedlings (E monandrumand B longifolia) were collected from the montane moist evergreen broad-leaved forestin the Ailao Mountains These seedlings were pre-incubated in tap water for two weekshence the nutrient content of different individuals was made relatively equal Sterilizedvermiculite was used as growth substrate with an equal volume of vermiculite (100 mL)poured into each cultivated pot three seedlings per pot Fertilizer was added in the form ofnutrient solutions 10-mL nutrient solutions were added to the culture pots every two daysIn August 2018 the leaf stem and root of each individual were collected separately andfirst washed with tap water then washed with distilled water and placed into envelopesand oven-dried to a constant weight (48 h) at 65 C These dried samples were placed in amortar containing liquid nitrogen and powdered using a pestle each powder sample wasplaced in a small Ziplock bag Because both stems and roots did not reach the minimummass required for element analysis only the element concentrations in leaf were measured

Table 1 The N and P concentrations and ratios in nutrient solutions used in treatments of greenhousecultivation experiment

Treatment N (mmol Lminus1) P (mmol Lminus1) N P

N1P3 05 025 2N2P3 1 025 4N3P3 2 025 8N4P3 4 025 16N5P3 6 025 24P1N3 2 00625 32P2N3 2 0125 16P4N3 2 05 4P5N3 2 075 27

Five N levels under moderate P level (P3) five P levels under moderate N level (N3)

23 Laboratory Analysis

The total C and N concentrations of all plant samples in the cultivation and fieldexperiments and soil samples in the field experiment were measured with a carbon andnitrogen element analyzer (Vario MAX CN Elemental Analyzer Elementar LangenselboldGermany) about 01 g dried powder of each plant and soil sample was used for themeasurement of C and N concentrations The P concentration of plant and soil sampleswere measured by an inductively coupled plasma atomic emission spectrometer (iCAP6300Thermo Fisher Scientific Waltham MA USA) after digested with HNO3-HClO4 For the

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determination of P concentrations of plant and soil samples 048ndash052 g and 01ndash012 gdried powder was needed respectively Total C N and P concentrations of both plant andhumus soil samples were calculated on per dry weight basis

24 Data Analysis and Statistics

The stoichiometric homeostasis index (H) of different organs of each plant species wascalculated according to the ecological stoichiometric homeostasis model [17]

y = cx1H (1)

where x refers to the N or P content or the ratio of NP in the growing substrate y refers tothe content of N or P or the NP ratio in a given plant organ c is a constant In this model avalue of H gt 1 may be interpreted to mean that the plant has the ability to regulate elementalcomposition [17] Persson et al [33] classified organisms into four types according to therange of their H values as follows H gt 4 strict homeostasis 2 lt H lt 4 weakly homeostasis34 lt H lt 2 weakly plastic H lt 34 plastic

In the field experiment the differences in the C N and P elements and their stoi-chiometric ratios of facultative epiphytes between epiphytic and terrestrial habitats wereanalyzed by independent t-test and two-way ANOVA The H for N P and NP of leavesstems and roots of three species were estimated through regression analysis Variation ofH across different species organs and elements in the field experiment was analyzed byone-way ANOVA and two-way ANOVA for which statistical significance was set at p =005 Data normality and homogeneity were checked through Shapiro-Wilk and Levenetests for the data not met the assumptions Kruskal-Wallis tests were used

In the cultivation experiment the H for N P and NP of leaves were estimated throughregression analysis Variation of N P and NP in leaves of two species across differentfertilization levels were analyzed by two-way ANOVA for which statistical significancewas set at p = 005 All statistical analyses were done using IBM SPSS Statistics 190 (SPSSInc Chicago IL USA)

3 Results31 Stoichiometric Patterns of C N P Concentrations and Their Ratios in Different Organs inTwo Habitats in the Field

The C concentration and CN were only affected by species the N and P concentrationsand the CP and NP ratios in leaves were significantly affected by species and habitatwhile the CP was also affected by the interaction between species (Table 2) The C and Nconcentration and the CN CP NP ratios in stems were significantly affected by specieswhile C and CN were also affected by habitat the P concentration was only affected byhabitat For roots the C and N concentration were significantly affected by habitat andspecies the CN and NP ratios were significantly affected by species and the interactionbetween habitat and species the P and CP was only affected by species

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Table 2 Effects of species habitats and their interactions on stoichiometric traits of C N and P indifferent organs of facultative epiphytes in field experiment

Organ VariableHabitat Species Habitat times Species

F df F df F df

Leaf C 367 1 93 63420 2 93 154 2 93N 456 1 93 9122 2 93 129 2 93P 1083 1 89 4399 2 89 301 2 89

CN 056 1 93 17012 2 93 110 2 93CP 773 1 89 9448 2 89 370 2 89NP 399 1 89 453 2 89 089 2 89

Stem C 706 1 57 20677 1 57 203 1 57N 143 1 57 9392 1 57 070 1 57P 1138 1 44 284 1 44 107 1 44

CN 440 1 57 8456 1 57 082 1 57CP 374 1 44 892 1 44 002 1 44NP 324 1 44 1039 1 44 225 1 44

Root C 2339 1 90 2188 2 90 193 2 90N 469 1 90 3430 2 90 075 2 90P 338 1 65 1360 2 65 081 2 65

CN 009 1 90 6297 2 90 373 2 90CP 010 1 65 903 2 65 072 2 65NP 000 1 65 434 2 65 320 2 65

p lt 005 p lt 001 F indicates F value df denotes degree of freedom

For C gracilis the difference in the leaf N concentration and P concentration of stemand root between the two habitats was significant (p lt 005) the P concentrations of leafand the rootrsquos C and N concentrations in the epiphytic habitat significantly exceeded thosein terrestrial habitat (p lt 001) while the CN and CP in leaf between the two habitatsshowed a reverse trend (Table S1) For E monandrum the C and P concentration in leaf andthe C and N concentration and the CN ratio in root in the epiphytic habitat were higherthan those in terrestrial habitat (Table S2) For B longifolia the C N and P concentrations inleaf and root in the epiphytic habitat were higher than those in terrestrial habitat but viceversa for the CN and CP in leaf (Table S3) At the species level the plasticity of elementcomposition in epiphytic individuals of the three species were relatively higher than that ofterrestrial individuals expect C in root and P in stem of C gracilis C and N in leaf and rootof E monandrum and C and N in leaf and root CN in leaf of B longifolia (Tables S1ndashS3)

32 Variation of Stoichiometric Homeostasis among Different Species Organs and Elements inthe Field

For leaves the HN of C gracilis was weaker than either that of E monandrum orB longifolia while the latter was characterized by the strongest N homeostasis (Figure 2Table S4) The HP of species was ranked in the order of E monandrum gt C gracilis gt Blongifolia In terms of HNP it was strongest in C gracilis followed by E monandrum beingweakest in B longifolia For stems with the exception of NP E monandrum had H valuesfor N and P that were each stronger than those characterizing C gracilis For roots exceptfor HN the H values for P and NP descended among species in the order of B longifoliagt C gracilis gt E monandrum Whereas the corresponding order for HN was B longifolia gtE monandrum gt C gracilis For the mean values of H the difference of leaves and rootsamong three species and stems between two species were all not significant (Figure S1)

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Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stemand (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrumB longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species allstronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stemsand roots the mean values for HN and HP of the three species were all decreased in theorder of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems thehomeostasis for NP was stronger than the P element but weaker than the N element Inroots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gtHN gt HP)

Forests 2021 12 x FOR PEER REVIEW 8 of 15

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratiosin leaves stems and roots of three species Bars are the mean values with standard error Capital andlowercase letters indicate significant differences among organs and element types respectively

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment

In the cultivation experiment the concentration of N and P in leaves were significantlyaffected by species and treatment levels NP was significantly affected by the treatment level

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while the interaction between species and treatment level had no significant effects on theN and P concentration and their elemental ratio (Table 3) The N and P concentrations ofleaves were both increased as higher N and P concentrations were applied in solution For thestoichiometric homeostasis values of different elements of leaves in the two species all valueswere greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the Nelement was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their interactionon N and P concentrations and NP ratios in leaves of two species in the cultivation experiment

VariableSpecies Treatment Species times Level

F df p F df p F df p

N 22611 1 000 417 8 003 132 8 022P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098The numerator df are given in the table the denominator df is 8

Forests 2021 12 x FOR PEER REVIEW 9 of 15

treatment level while the interaction between species and treatment level had no signif-icant effects on the N and P concentration and their elemental ratio (Table 3) The N and P concentrations of leaves were both increased as higher N and P concentrations were ap-plied in solution For the stoichiometric homeostasis values of different elements of leaves in the two species all values were greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the N element was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their in-teraction on N and P concentrations and NP ratios in leaves of two species in the cultivation ex-periment

Variable Species Treatment Species times Level

F df p F df p F df p N 22611 1 000 417 8 003 132 8 022 P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098 The numerator df are given in the table the denominator df is 8

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N P and NP in the greenhouse cultivation experiment (a b c) H values for leaf N P and N P of Briggsia longifolia (d e f) H values for leaf N P and N P of Elatostema monandrum respectively

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N Pand NP in the greenhouse cultivation experiment (andashc) H values for leaf N P and N P of Briggsialongifolia (dndashf) H values for leaf N P and N P of Elatostema monandrum respectively

Forests 2021 12 16 10 of 15

4 Discussion41 Differences in Nutrient Use Strategy between Epiphytic and Terrestrial Individuals ofFacultative Epiphytes

In our field experiment the concentrations of N and P in epiphytic individuals ofthree species in the natural forest were higher than those in terrestrial individuals but CNCP and NP ratios in the former were lower than the latter (Tables S1ndashS3) This agreedwith the results from fern in our study area and bromeliads in humid montane forest andlowland forests elsewhere [134748] CN and CP ratios reflect the carbon fixation per unitnutrient the higher N and P concentration and lower CN and CP values are associatedwith luxury consumption of plants [164950] Accordingly in our study the use of N andP by epiphytic individuals were more luxurious than terrestrial individuals which agreeswith the result that terrestrial individuals of fern are more efficient in nutrient use thanepiphytic individuals [13] The plasticity in nutrient use strategy of facultative epiphytes isconsidered to be an adaptive mechanism to different environmental conditions in orderto exploit the available resources efficiently [1213] Our results indicated that facultativeepiphytes had more flexible plasticity in nutrient use strategies to adapt habitat shifts

Winkler and Zotz [51] found that epiphytes possessed lower nutrient use efficiencythan that of terrestrial plants Epiphytes are generally considered to survive in the nutrientintermittent epiphytic habitats through luxurious consumption of nutrients [12] Theyusually absorb large amounts of nutrients and store them in vacuoles when the nutrient isabundant [142949] Accordingly the lower N and P use of epiphytic individuals may berelated to the luxury uptake of N and P in epiphytic habitats Furthermore we found thatvariation in the elemental composition of epiphytic individuals exceeded that of terrestrialindividuals most likely because epiphytic habitats are more heterogeneous [552] and theelemental composition of plants is influenced by other environmental factors (eg wateravailability light intensity) besides soil alone [3849]

42 Effects of Nutrient Limitation on the Distribution of Three Facultative Epiphytes

The foliar NP ratio can be used as an indicator of the type of nutrient limitationon plant growth the lower NP ratios indicate the plants were limited by N [244953]whereas the higher NP ratios indicates P limitations were present [2954] Epiphytes areusually restricted by P [55] but in our research nutrient restriction types differ amongthe three species The NP ratio of terrestrial and epiphytic individuals of E monandrumand epiphytic individuals of B longifolia was lower than 12 (p-values = 0214 0526 0225respectively) (Tables S2 and S3) suggesting the growth of which was restricted by Navailability [29] Since the NP ratio in terrestrial and epiphytic individuals of C gracilisand terrestrial individuals of B longifolia was higher than 12 (p-values = 0004 0227 0009respectively) yet still lower than 16 (p-values = 0 005 0 0006 respectively) (Tables S1 andS3) we interpreted this to mean that the growth of which were limited by N or co-limited byN and P [28] Our results were consistent with the finding that facultative epiphytes on datepalms (Phoenix dactylifera) in southeastern Spain were subject to N-limited conditions [14]Furthermore in our research the lower NP ratio in epiphytic than terrestrial individualsimplied that N-restriction in the epiphytic habitat was more pronounced The higher Pconcentration in epiphytic individuals probably increases the demands for N thus makingthem more pronounced Furthermore the higher P concentration in plants can alleviate thedamage caused by drought stress [49] In this study the higher P concentration in epiphyticindividuals may reflect their adaptation to epiphytic habitats where water is intermittent

The factors that drive the distribution of facultative epiphytes are not clear butprevious results suggested that environmental factors can affect the distribution of epi-phytes [1446] For the three species in our research most individuals of E monandrum andB longifolia grow on the bark with mass mat or thick humus while the majority of C gracilisgrow on the forest floor For E monandrum and B longifolia the N-restriction in epiphytichabitats was stronger than terrestrial habitats therefore they may gradually shift to forestfloor to alleviate N limitation For C gracilis the type of nutrient limitation in terrestrial and

Forests 2021 12 16 11 of 15

epiphytic habitats was same this indicated that the nutrient status in epiphytic habitatscan satisfy its requirement Previous research indicated that the interspecific competition interrestrial habitats was more intense than that in epiphytic habitats [7] Therefore C gracilismay shift to epiphytic habitats to escape interspecific competition

43 Differences in Stoichiometric Homeostasis among Species Organs and Elements ofFacultative Epiphytes

Traditional theory predicts that the stoichiometric homeostasis of autotroph was rela-tively weak [1756] However mounting data suggest that plants have a strong regulatorycapacity for their element composition [202237] Our results of the field and greenhousecultivation experiment indicated that the values of HN HP and HNP of leaves were allgt4 (p = 0002 0004 respectively) (Figure 4 Table S4) both showed strong homeostasiswhich did not support our second hypothesis that the homeostasis of facultative epiphytesis weak These results for facultative epiphytes in our study are also at odds with previousresearch concluded that a weak homeostasis of plants implied higher ecological adapt-ability and better suitability to a more variable environment [33] This discrepancy maybe because that conclusion about relationship between stoichiometric homeostasis andecological adaptability of plants was almost based on studies of plant leaves [3349] Theelemental composition of leaves is insensitive to nutrient changes in the soil instead rootsare more valid indicators of nutrient change in the environment [203857ndash59] Therefore aleafrsquos stoichiometric homeostasis cannot accurately reflect the ecological adaptability ofplants [3860] In our work here although leaves did have strong stoichiometric home-ostasis it was rather weak for roots (Table S4) The main function of roots is to absorbmineral nutrients and water from the soil [33] their plastic nutrient absorption is helpfulfor facultative epiphytes to efficiently exploit the available resources in different habitatsThis result indicated that facultative epiphytes could adapt to variable environment bychanging the elemental composition of their roots

Moreover the difference in stoichiometric homeostasis among the three species in ourfield study was not significant and the stoichiometric homeostasis mainly affected by or-gans (Table S5) likely because all three share a similar species richness in the sampling areaand their ecological strategy and adaptability to the environment were comparable [22]This was also confirmed by the consistent response of the two species to nutrient additionin the greenhouse cultivation experiments in our study (Table 3)

Different organs of the same species exhibit disparate stoichiometric characteristicsto better adapt to the environment [162033] We found that stoichiometric homeostasiswas stronger in aboveground than belowground of the three facultative epiphytes Rootswere more sensitive to changes of elements concentrations in environmental resourceswhich agrees with findings reported for terrestrial grass and woody plants [21223138]However unlike the related studies on terrestrial woody plants [38] the stems of C gracilisand E monandrum show strong stoichiometric homeostasis (Table S4) This may be relatedto the insensitivity of some key elements in stems of facultative epiphyte to changes insoil nutrient concentrations The stems of woody and herbaceous plants take differentfunctions the stems of woody plants mainly play transportation and supporting rolewhile the stems of herbaceous plants have less structural C than woody plants yet bearthe functions of photosynthesis transportation and the supporting action [20] Thereforethe stems of herbaceous plants need relatively stable supply of elements to maintain theirphysiological functions

The strength of homeostasis is also related to the elemental concentration [61] Previ-ous research for terrestrial plants indicated that the homeostasis of macro-elements washigher than trace elements [3461] In our study the value of stoichiometric homeostasisof N and P in the three facultative epiphyte species showed the order of HN gt HP This isconsistent with results from vertebrates and trees with greater control over high-demandelements [34ndash36] Therefore the facultative epiphytic plants may also have greater con-trol over macro-elements highly demanded for physiology functioning However ourresearch only focused on the stoichiometric homeostasis of N and P elements in facultative

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epiphytes the stoichiometric homeostasis of trace elements remains unknown Thereforewhether the stoichiometric homeostasis of macro-elements in facultative epiphytes is higherthan trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumulationof N within a given plant tissue is usually accompanied by an increase in its P content thehomeostasis of NP is stronger than N and P elements [1762] However in our study thestoichiometric homeostasis value of NP in leaf and root were all higher than that of P butlower than that of N (Figure 3) Based on the theory that limiting elements should havestrong homeostasis [313463] we speculate that facultative epiphytes may be restricted byN availability These plants therefore would take strong regulation of N to cope with theunpredictable fluctuations of N elements in their habitat

5 Conclusions

This study systematically elucidates the nutrient use and ecological adaptation strate-gies of facultative epiphytes from the perspective of stoichiometry using field investigationand greenhouse cultivation experiments Our study showed that epiphytic individualsof facultative epiphytes survive intermittent habitat via luxury consumption of nutrientswhile terrestrial individuals use more conservative nutrients (Figure 5) The nutrient usestrategies of facultative epiphytes were flexible which enabled facultative epiphytes toexploit bark and soil interchangeably Facultative epiphytes had strong control over N andP concentrations and NP ratio of leaves and stems while roots had weaker stoichiometricstability These findings suggested that to adapt to the variable habitats in montane forestecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulatingthe absorption and composition of elements in roots

|

Forests 2021 12 x FOR PEER REVIEW 12 of 15

trol over macro-elements highly demanded for physiology functioning However our research only focused on the stoichiometric homeostasis of N and P elements in faculta-tive epiphytes the stoichiometric homeostasis of trace elements remains unknown Therefore whether the stoichiometric homeostasis of macro-elements in facultative epi-phytes is higher than trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumu-lation of N within a given plant tissue is usually accompanied by an increase in its P content the homeostasis of NP is stronger than N and P elements [1762] However in our study the stoichiometric homeostasis value of NP in leaf and root were all higher than that of P but lower than that of N (Figure 3) Based on the theory that limiting ele-ments should have strong homeostasis [313463] we speculate that facultative epiphytes may be restricted by N availability These plants therefore would take strong regulation of N to cope with the unpredictable fluctuations of N elements in their habitat

5 Conclusions This study systematically elucidates the nutrient use and ecological adaptation

strategies of facultative epiphytes from the perspective of stoichiometry using field in-vestigation and greenhouse cultivation experiments Our study showed that epiphytic individuals of facultative epiphytes survive intermittent habitat via luxury consumption of nutrients while terrestrial individuals use more conservative nutrients (Figure 5) The nutrient use strategies of facultative epiphytes were flexible which enabled facultative epiphytes to exploit bark and soil interchangeably Facultative epiphytes had strong control over N and P concentrations and NP ratio of leaves and stems while roots had weaker stoichiometric stability These findings suggested that to adapt to the variable habitats in montane forest ecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulating the absorption and composition of elements in roots

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic and terrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at wwwmdpicomxxxs1 Fig-ure S1 The mean stoichiometric homeostasis (H) values for N and P elements and their element ratios in leaf stem and root of different species Table S1 The difference of stoichiometric traits of C N and P concentrations and their element ratios in different organs of C gracilis between epi-

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic andterrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at httpswwwmdpicom1999-490712116s1 Figure S1 The mean stoichiometric homeostasis (H) values for N and Pelements and their element ratios in leaf stem and root of different species Table S1 The differenceof stoichiometric traits of C N and P concentrations and their element ratios in different organs of C

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a low nutrient use efficiency [14] However whether epiphytic individuals of facultativeepiphytes (which occur both epiphytically and terrestrially) are more efficient in nutrientuse than terrestrial individuals remain an open question In this research to study theecological stoichiometric characteristics and homeostasis of C N and P in three commonvascular facultative epiphytes in the montane moist evergreen broad-leaved forest of AilaoMountain National Nature Reserve we conducted a field experiment and a greenhousecultivation experiment We proposed the following hypotheses (1) There are significantdifferences in adaptation strategy between the epiphytic and terrestrial individuals offacultative epiphytes which reflect in their stoichiometry pattern (2) Facultative epiphytesexhibit weak stoichiometric homeostasis to adapt to variable habitats (3) and they showstronger control on the elemental composition of leaves than that of roots

2 Materials and Methods21 Study Area and Species

This study was conducted in subtropical montane moist evergreen broad-leaved forestin Xujiaba the core area of the Ailao Mountain National Natural Reserve Southwest China(2336primendash2456prime N 10044primendash10130prime E) The Ailao Mountain have the largest most intactmountain moist evergreen broad-leaved forest in China forming an important portion ofthis countryrsquos subtropical forest ecosystem [44] In this montane moist evergreen broad-leaved forest the canopy and trunk are rich in epiphytic plant communities [43] providinga natural and ideal place to study the stoichiometric characteristics of epiphytes The AilaoMountain are located in a climate transition area in the central and northern parts ofYunnanrsquos subtropical zone characterized by dry and rainy seasons due to the influence ofthe southwest monsoon According to long-term monitoring data from the Ailao MountainEcological Station of the Chinese Academy of Sciences the annual precipitation in this areais 1947 mm with an annual evaporation of 1192 mm and the dry season (NovemberndashApril)and rainy season (MayndashOctober) are distinct The precipitation during the rainy seasonaccounts for ca 85 of the annual precipitation at the site where the relative humidity is85 annual average temperature is 113 C monthly average maximum temperature is157 C and monthly average minimum temperature is 56 C [45]

Xujiaba area is rich in epiphytes there are nearly 600 species including lichens (183)bryophytes (176) ferns (117) and spermatophytes (113) [843] The dominant facultative epi-phytic spermatophytes are Cautleya gracilis (Smith) Dandy (an annual erect herb 025ndash08 min height) Elatostema monandrum (D Don) Hara (a small annual herb 005ndash02 m in height)and Briggsia longifolia var multiflora (a perennial herb without stem) [43] In this areaaccording to our survey epiphytic individuals of facultative epiphytes are usually foundon tree trunks with moss mat or thicker humus soil while terrestrial individuals are usuallydistributed in the forest floor under canopy gap (Figure 1) Most individuals of C gracilisgrow on the forest floor while E monandrum and B longifolia show the inverse pattern Thedistribution of facultative epiphytes may be affected by the seed supply and microclimaticconditions [46]

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Figure 1 The pictures of investigated species (AB) epiphytic and terrestrial individuals of Cautleya gracilis (CD) epiphytic and terrestrial individuals of Elatostema monandrum (EF) epi-phytic and terrestrial individuals of Briggsia longifolia respectively

22 Field Sampling and Greenhouse Culture In the field sampling experiment we used the natural nutrient gradients of the forest

canopy and ground soil stands to carry out nutrient additions Many factors such as light substrate water and nutrient content jointly affect the content of chemical elements in plants in the field [3234] so the relationship between a plantrsquos elemental content and its growth substrate cannot be accurately estimated by field sampling alone Therefore to accurately evaluate the stoichiometric stability of facultative epiphytes we also per-formed a greenhouse cultivation experiment in addition to the field experiment

Field sampling was done during the rainy season (August 2018) Four collection line transects were set along four evenly divided directions in the core area of montane moist evergreen broad-leaved forest in the Ailao Mountains and 3ndash5 sampling points were selected per line transect We defined individuals that grow on the trunk as epiphytic individuals and individuals that grow on the ground as terrestrial individuals The healthy mature terrestrial and epiphytic individuals of three dominant facultative epi-phytes (C gracilis E monandrum and B longifolia) were separately collected from each sampling point for which terrestrial and epiphytic individuals were collected from the forests soil and c 15 m of their host trees respectively In total 34 samples (16 samples from epiphytic individuals and 18 samples from terrestrial individuals) were collected

Figure 1 The pictures of investigated species (AB) epiphytic and terrestrial individuals of Cautleya gracilis (CD)epiphytic and terrestrial individuals of Elatostema monandrum (EF) epiphytic and terrestrial individuals of Briggsia longifoliarespectively

22 Field Sampling and Greenhouse Culture

In the field sampling experiment we used the natural nutrient gradients of the forestcanopy and ground soil stands to carry out nutrient additions Many factors such as lightsubstrate water and nutrient content jointly affect the content of chemical elements inplants in the field [3234] so the relationship between a plantrsquos elemental content and itsgrowth substrate cannot be accurately estimated by field sampling alone Therefore toaccurately evaluate the stoichiometric stability of facultative epiphytes we also performeda greenhouse cultivation experiment in addition to the field experiment

Field sampling was done during the rainy season (August 2018) Four collectionline transects were set along four evenly divided directions in the core area of montanemoist evergreen broad-leaved forest in the Ailao Mountains and 3ndash5 sampling points wereselected per line transect We defined individuals that grow on the trunk as epiphyticindividuals and individuals that grow on the ground as terrestrial individuals Thehealthy mature terrestrial and epiphytic individuals of three dominant facultative epiphytes(C gracilis E monandrum and B longifolia) were separately collected from each sampling

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point for which terrestrial and epiphytic individuals were collected from the forests soiland c 15 m of their host trees respectively In total 34 samples (16 samples from epiphyticindividuals and 18 samples from terrestrial individuals) were collected for C gracilis 30samples (18 samples from epiphytic individuals and 12 samples from terrestrial individuals)for E monandrum and 38 samples (20 samples from epiphytic individuals and 18 samplesfrom terrestrial individuals) for B longifolia We collected whole plant individuals whoseroots stems and leaves were then separated and stored as well the soil at their growthpositions of the target species for use a growing substrate for these plants These plantsamples were first washed with tap water then washed with distilled water and placed intoenvelopes and oven-dried to a constant weight (48 h) at 65 C then the dried samples wereground to powder The soil samples were dried ground and passed through a 60-meshsieve before their analysis

The cultivation experiment was carried out in a greenhouse from April to August2018 In this experiment the N and P fertilizers used were respectively NH4NO3 andNaH2PO4 their fertilization gradients are shown in Table 1 which consisted of ninetreatments in total with three replicates per treatment There were five N levels (05 1 24 and 6 mmol Lminus1) under a moderate P gradient (025 mmol Lminus1) and five P gradients(00625 0125 025 05 and 075 mmol Lminus1) under a moderate N gradient (2 mmol Lminus1) Itis difficult to collect and cultivate seedlings of C gracilis thus only the other two specieswere used for the cultivation experiment Healthy epiphytic seedlings (E monandrumand B longifolia) were collected from the montane moist evergreen broad-leaved forestin the Ailao Mountains These seedlings were pre-incubated in tap water for two weekshence the nutrient content of different individuals was made relatively equal Sterilizedvermiculite was used as growth substrate with an equal volume of vermiculite (100 mL)poured into each cultivated pot three seedlings per pot Fertilizer was added in the form ofnutrient solutions 10-mL nutrient solutions were added to the culture pots every two daysIn August 2018 the leaf stem and root of each individual were collected separately andfirst washed with tap water then washed with distilled water and placed into envelopesand oven-dried to a constant weight (48 h) at 65 C These dried samples were placed in amortar containing liquid nitrogen and powdered using a pestle each powder sample wasplaced in a small Ziplock bag Because both stems and roots did not reach the minimummass required for element analysis only the element concentrations in leaf were measured

Table 1 The N and P concentrations and ratios in nutrient solutions used in treatments of greenhousecultivation experiment

Treatment N (mmol Lminus1) P (mmol Lminus1) N P

N1P3 05 025 2N2P3 1 025 4N3P3 2 025 8N4P3 4 025 16N5P3 6 025 24P1N3 2 00625 32P2N3 2 0125 16P4N3 2 05 4P5N3 2 075 27

Five N levels under moderate P level (P3) five P levels under moderate N level (N3)

23 Laboratory Analysis

The total C and N concentrations of all plant samples in the cultivation and fieldexperiments and soil samples in the field experiment were measured with a carbon andnitrogen element analyzer (Vario MAX CN Elemental Analyzer Elementar LangenselboldGermany) about 01 g dried powder of each plant and soil sample was used for themeasurement of C and N concentrations The P concentration of plant and soil sampleswere measured by an inductively coupled plasma atomic emission spectrometer (iCAP6300Thermo Fisher Scientific Waltham MA USA) after digested with HNO3-HClO4 For the

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determination of P concentrations of plant and soil samples 048ndash052 g and 01ndash012 gdried powder was needed respectively Total C N and P concentrations of both plant andhumus soil samples were calculated on per dry weight basis

24 Data Analysis and Statistics

The stoichiometric homeostasis index (H) of different organs of each plant species wascalculated according to the ecological stoichiometric homeostasis model [17]

y = cx1H (1)

where x refers to the N or P content or the ratio of NP in the growing substrate y refers tothe content of N or P or the NP ratio in a given plant organ c is a constant In this model avalue of H gt 1 may be interpreted to mean that the plant has the ability to regulate elementalcomposition [17] Persson et al [33] classified organisms into four types according to therange of their H values as follows H gt 4 strict homeostasis 2 lt H lt 4 weakly homeostasis34 lt H lt 2 weakly plastic H lt 34 plastic

In the field experiment the differences in the C N and P elements and their stoi-chiometric ratios of facultative epiphytes between epiphytic and terrestrial habitats wereanalyzed by independent t-test and two-way ANOVA The H for N P and NP of leavesstems and roots of three species were estimated through regression analysis Variation ofH across different species organs and elements in the field experiment was analyzed byone-way ANOVA and two-way ANOVA for which statistical significance was set at p =005 Data normality and homogeneity were checked through Shapiro-Wilk and Levenetests for the data not met the assumptions Kruskal-Wallis tests were used

In the cultivation experiment the H for N P and NP of leaves were estimated throughregression analysis Variation of N P and NP in leaves of two species across differentfertilization levels were analyzed by two-way ANOVA for which statistical significancewas set at p = 005 All statistical analyses were done using IBM SPSS Statistics 190 (SPSSInc Chicago IL USA)

3 Results31 Stoichiometric Patterns of C N P Concentrations and Their Ratios in Different Organs inTwo Habitats in the Field

The C concentration and CN were only affected by species the N and P concentrationsand the CP and NP ratios in leaves were significantly affected by species and habitatwhile the CP was also affected by the interaction between species (Table 2) The C and Nconcentration and the CN CP NP ratios in stems were significantly affected by specieswhile C and CN were also affected by habitat the P concentration was only affected byhabitat For roots the C and N concentration were significantly affected by habitat andspecies the CN and NP ratios were significantly affected by species and the interactionbetween habitat and species the P and CP was only affected by species

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Table 2 Effects of species habitats and their interactions on stoichiometric traits of C N and P indifferent organs of facultative epiphytes in field experiment

Organ VariableHabitat Species Habitat times Species

F df F df F df

Leaf C 367 1 93 63420 2 93 154 2 93N 456 1 93 9122 2 93 129 2 93P 1083 1 89 4399 2 89 301 2 89

CN 056 1 93 17012 2 93 110 2 93CP 773 1 89 9448 2 89 370 2 89NP 399 1 89 453 2 89 089 2 89

Stem C 706 1 57 20677 1 57 203 1 57N 143 1 57 9392 1 57 070 1 57P 1138 1 44 284 1 44 107 1 44

CN 440 1 57 8456 1 57 082 1 57CP 374 1 44 892 1 44 002 1 44NP 324 1 44 1039 1 44 225 1 44

Root C 2339 1 90 2188 2 90 193 2 90N 469 1 90 3430 2 90 075 2 90P 338 1 65 1360 2 65 081 2 65

CN 009 1 90 6297 2 90 373 2 90CP 010 1 65 903 2 65 072 2 65NP 000 1 65 434 2 65 320 2 65

p lt 005 p lt 001 F indicates F value df denotes degree of freedom

For C gracilis the difference in the leaf N concentration and P concentration of stemand root between the two habitats was significant (p lt 005) the P concentrations of leafand the rootrsquos C and N concentrations in the epiphytic habitat significantly exceeded thosein terrestrial habitat (p lt 001) while the CN and CP in leaf between the two habitatsshowed a reverse trend (Table S1) For E monandrum the C and P concentration in leaf andthe C and N concentration and the CN ratio in root in the epiphytic habitat were higherthan those in terrestrial habitat (Table S2) For B longifolia the C N and P concentrations inleaf and root in the epiphytic habitat were higher than those in terrestrial habitat but viceversa for the CN and CP in leaf (Table S3) At the species level the plasticity of elementcomposition in epiphytic individuals of the three species were relatively higher than that ofterrestrial individuals expect C in root and P in stem of C gracilis C and N in leaf and rootof E monandrum and C and N in leaf and root CN in leaf of B longifolia (Tables S1ndashS3)

32 Variation of Stoichiometric Homeostasis among Different Species Organs and Elements inthe Field

For leaves the HN of C gracilis was weaker than either that of E monandrum orB longifolia while the latter was characterized by the strongest N homeostasis (Figure 2Table S4) The HP of species was ranked in the order of E monandrum gt C gracilis gt Blongifolia In terms of HNP it was strongest in C gracilis followed by E monandrum beingweakest in B longifolia For stems with the exception of NP E monandrum had H valuesfor N and P that were each stronger than those characterizing C gracilis For roots exceptfor HN the H values for P and NP descended among species in the order of B longifoliagt C gracilis gt E monandrum Whereas the corresponding order for HN was B longifolia gtE monandrum gt C gracilis For the mean values of H the difference of leaves and rootsamong three species and stems between two species were all not significant (Figure S1)

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Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stemand (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrumB longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species allstronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stemsand roots the mean values for HN and HP of the three species were all decreased in theorder of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems thehomeostasis for NP was stronger than the P element but weaker than the N element Inroots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gtHN gt HP)

Forests 2021 12 x FOR PEER REVIEW 8 of 15

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratiosin leaves stems and roots of three species Bars are the mean values with standard error Capital andlowercase letters indicate significant differences among organs and element types respectively

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment

In the cultivation experiment the concentration of N and P in leaves were significantlyaffected by species and treatment levels NP was significantly affected by the treatment level

Forests 2021 12 16 9 of 15

while the interaction between species and treatment level had no significant effects on theN and P concentration and their elemental ratio (Table 3) The N and P concentrations ofleaves were both increased as higher N and P concentrations were applied in solution For thestoichiometric homeostasis values of different elements of leaves in the two species all valueswere greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the Nelement was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their interactionon N and P concentrations and NP ratios in leaves of two species in the cultivation experiment

VariableSpecies Treatment Species times Level

F df p F df p F df p

N 22611 1 000 417 8 003 132 8 022P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098The numerator df are given in the table the denominator df is 8

Forests 2021 12 x FOR PEER REVIEW 9 of 15

treatment level while the interaction between species and treatment level had no signif-icant effects on the N and P concentration and their elemental ratio (Table 3) The N and P concentrations of leaves were both increased as higher N and P concentrations were ap-plied in solution For the stoichiometric homeostasis values of different elements of leaves in the two species all values were greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the N element was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their in-teraction on N and P concentrations and NP ratios in leaves of two species in the cultivation ex-periment

Variable Species Treatment Species times Level

F df p F df p F df p N 22611 1 000 417 8 003 132 8 022 P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098 The numerator df are given in the table the denominator df is 8

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N P and NP in the greenhouse cultivation experiment (a b c) H values for leaf N P and N P of Briggsia longifolia (d e f) H values for leaf N P and N P of Elatostema monandrum respectively

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N Pand NP in the greenhouse cultivation experiment (andashc) H values for leaf N P and N P of Briggsialongifolia (dndashf) H values for leaf N P and N P of Elatostema monandrum respectively

Forests 2021 12 16 10 of 15

4 Discussion41 Differences in Nutrient Use Strategy between Epiphytic and Terrestrial Individuals ofFacultative Epiphytes

In our field experiment the concentrations of N and P in epiphytic individuals ofthree species in the natural forest were higher than those in terrestrial individuals but CNCP and NP ratios in the former were lower than the latter (Tables S1ndashS3) This agreedwith the results from fern in our study area and bromeliads in humid montane forest andlowland forests elsewhere [134748] CN and CP ratios reflect the carbon fixation per unitnutrient the higher N and P concentration and lower CN and CP values are associatedwith luxury consumption of plants [164950] Accordingly in our study the use of N andP by epiphytic individuals were more luxurious than terrestrial individuals which agreeswith the result that terrestrial individuals of fern are more efficient in nutrient use thanepiphytic individuals [13] The plasticity in nutrient use strategy of facultative epiphytes isconsidered to be an adaptive mechanism to different environmental conditions in orderto exploit the available resources efficiently [1213] Our results indicated that facultativeepiphytes had more flexible plasticity in nutrient use strategies to adapt habitat shifts

Winkler and Zotz [51] found that epiphytes possessed lower nutrient use efficiencythan that of terrestrial plants Epiphytes are generally considered to survive in the nutrientintermittent epiphytic habitats through luxurious consumption of nutrients [12] Theyusually absorb large amounts of nutrients and store them in vacuoles when the nutrient isabundant [142949] Accordingly the lower N and P use of epiphytic individuals may berelated to the luxury uptake of N and P in epiphytic habitats Furthermore we found thatvariation in the elemental composition of epiphytic individuals exceeded that of terrestrialindividuals most likely because epiphytic habitats are more heterogeneous [552] and theelemental composition of plants is influenced by other environmental factors (eg wateravailability light intensity) besides soil alone [3849]

42 Effects of Nutrient Limitation on the Distribution of Three Facultative Epiphytes

The foliar NP ratio can be used as an indicator of the type of nutrient limitationon plant growth the lower NP ratios indicate the plants were limited by N [244953]whereas the higher NP ratios indicates P limitations were present [2954] Epiphytes areusually restricted by P [55] but in our research nutrient restriction types differ amongthe three species The NP ratio of terrestrial and epiphytic individuals of E monandrumand epiphytic individuals of B longifolia was lower than 12 (p-values = 0214 0526 0225respectively) (Tables S2 and S3) suggesting the growth of which was restricted by Navailability [29] Since the NP ratio in terrestrial and epiphytic individuals of C gracilisand terrestrial individuals of B longifolia was higher than 12 (p-values = 0004 0227 0009respectively) yet still lower than 16 (p-values = 0 005 0 0006 respectively) (Tables S1 andS3) we interpreted this to mean that the growth of which were limited by N or co-limited byN and P [28] Our results were consistent with the finding that facultative epiphytes on datepalms (Phoenix dactylifera) in southeastern Spain were subject to N-limited conditions [14]Furthermore in our research the lower NP ratio in epiphytic than terrestrial individualsimplied that N-restriction in the epiphytic habitat was more pronounced The higher Pconcentration in epiphytic individuals probably increases the demands for N thus makingthem more pronounced Furthermore the higher P concentration in plants can alleviate thedamage caused by drought stress [49] In this study the higher P concentration in epiphyticindividuals may reflect their adaptation to epiphytic habitats where water is intermittent

The factors that drive the distribution of facultative epiphytes are not clear butprevious results suggested that environmental factors can affect the distribution of epi-phytes [1446] For the three species in our research most individuals of E monandrum andB longifolia grow on the bark with mass mat or thick humus while the majority of C gracilisgrow on the forest floor For E monandrum and B longifolia the N-restriction in epiphytichabitats was stronger than terrestrial habitats therefore they may gradually shift to forestfloor to alleviate N limitation For C gracilis the type of nutrient limitation in terrestrial and

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epiphytic habitats was same this indicated that the nutrient status in epiphytic habitatscan satisfy its requirement Previous research indicated that the interspecific competition interrestrial habitats was more intense than that in epiphytic habitats [7] Therefore C gracilismay shift to epiphytic habitats to escape interspecific competition

43 Differences in Stoichiometric Homeostasis among Species Organs and Elements ofFacultative Epiphytes

Traditional theory predicts that the stoichiometric homeostasis of autotroph was rela-tively weak [1756] However mounting data suggest that plants have a strong regulatorycapacity for their element composition [202237] Our results of the field and greenhousecultivation experiment indicated that the values of HN HP and HNP of leaves were allgt4 (p = 0002 0004 respectively) (Figure 4 Table S4) both showed strong homeostasiswhich did not support our second hypothesis that the homeostasis of facultative epiphytesis weak These results for facultative epiphytes in our study are also at odds with previousresearch concluded that a weak homeostasis of plants implied higher ecological adapt-ability and better suitability to a more variable environment [33] This discrepancy maybe because that conclusion about relationship between stoichiometric homeostasis andecological adaptability of plants was almost based on studies of plant leaves [3349] Theelemental composition of leaves is insensitive to nutrient changes in the soil instead rootsare more valid indicators of nutrient change in the environment [203857ndash59] Therefore aleafrsquos stoichiometric homeostasis cannot accurately reflect the ecological adaptability ofplants [3860] In our work here although leaves did have strong stoichiometric home-ostasis it was rather weak for roots (Table S4) The main function of roots is to absorbmineral nutrients and water from the soil [33] their plastic nutrient absorption is helpfulfor facultative epiphytes to efficiently exploit the available resources in different habitatsThis result indicated that facultative epiphytes could adapt to variable environment bychanging the elemental composition of their roots

Moreover the difference in stoichiometric homeostasis among the three species in ourfield study was not significant and the stoichiometric homeostasis mainly affected by or-gans (Table S5) likely because all three share a similar species richness in the sampling areaand their ecological strategy and adaptability to the environment were comparable [22]This was also confirmed by the consistent response of the two species to nutrient additionin the greenhouse cultivation experiments in our study (Table 3)

Different organs of the same species exhibit disparate stoichiometric characteristicsto better adapt to the environment [162033] We found that stoichiometric homeostasiswas stronger in aboveground than belowground of the three facultative epiphytes Rootswere more sensitive to changes of elements concentrations in environmental resourceswhich agrees with findings reported for terrestrial grass and woody plants [21223138]However unlike the related studies on terrestrial woody plants [38] the stems of C gracilisand E monandrum show strong stoichiometric homeostasis (Table S4) This may be relatedto the insensitivity of some key elements in stems of facultative epiphyte to changes insoil nutrient concentrations The stems of woody and herbaceous plants take differentfunctions the stems of woody plants mainly play transportation and supporting rolewhile the stems of herbaceous plants have less structural C than woody plants yet bearthe functions of photosynthesis transportation and the supporting action [20] Thereforethe stems of herbaceous plants need relatively stable supply of elements to maintain theirphysiological functions

The strength of homeostasis is also related to the elemental concentration [61] Previ-ous research for terrestrial plants indicated that the homeostasis of macro-elements washigher than trace elements [3461] In our study the value of stoichiometric homeostasisof N and P in the three facultative epiphyte species showed the order of HN gt HP This isconsistent with results from vertebrates and trees with greater control over high-demandelements [34ndash36] Therefore the facultative epiphytic plants may also have greater con-trol over macro-elements highly demanded for physiology functioning However ourresearch only focused on the stoichiometric homeostasis of N and P elements in facultative

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epiphytes the stoichiometric homeostasis of trace elements remains unknown Thereforewhether the stoichiometric homeostasis of macro-elements in facultative epiphytes is higherthan trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumulationof N within a given plant tissue is usually accompanied by an increase in its P content thehomeostasis of NP is stronger than N and P elements [1762] However in our study thestoichiometric homeostasis value of NP in leaf and root were all higher than that of P butlower than that of N (Figure 3) Based on the theory that limiting elements should havestrong homeostasis [313463] we speculate that facultative epiphytes may be restricted byN availability These plants therefore would take strong regulation of N to cope with theunpredictable fluctuations of N elements in their habitat

5 Conclusions

This study systematically elucidates the nutrient use and ecological adaptation strate-gies of facultative epiphytes from the perspective of stoichiometry using field investigationand greenhouse cultivation experiments Our study showed that epiphytic individualsof facultative epiphytes survive intermittent habitat via luxury consumption of nutrientswhile terrestrial individuals use more conservative nutrients (Figure 5) The nutrient usestrategies of facultative epiphytes were flexible which enabled facultative epiphytes toexploit bark and soil interchangeably Facultative epiphytes had strong control over N andP concentrations and NP ratio of leaves and stems while roots had weaker stoichiometricstability These findings suggested that to adapt to the variable habitats in montane forestecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulatingthe absorption and composition of elements in roots

|

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trol over macro-elements highly demanded for physiology functioning However our research only focused on the stoichiometric homeostasis of N and P elements in faculta-tive epiphytes the stoichiometric homeostasis of trace elements remains unknown Therefore whether the stoichiometric homeostasis of macro-elements in facultative epi-phytes is higher than trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumu-lation of N within a given plant tissue is usually accompanied by an increase in its P content the homeostasis of NP is stronger than N and P elements [1762] However in our study the stoichiometric homeostasis value of NP in leaf and root were all higher than that of P but lower than that of N (Figure 3) Based on the theory that limiting ele-ments should have strong homeostasis [313463] we speculate that facultative epiphytes may be restricted by N availability These plants therefore would take strong regulation of N to cope with the unpredictable fluctuations of N elements in their habitat

5 Conclusions This study systematically elucidates the nutrient use and ecological adaptation

strategies of facultative epiphytes from the perspective of stoichiometry using field in-vestigation and greenhouse cultivation experiments Our study showed that epiphytic individuals of facultative epiphytes survive intermittent habitat via luxury consumption of nutrients while terrestrial individuals use more conservative nutrients (Figure 5) The nutrient use strategies of facultative epiphytes were flexible which enabled facultative epiphytes to exploit bark and soil interchangeably Facultative epiphytes had strong control over N and P concentrations and NP ratio of leaves and stems while roots had weaker stoichiometric stability These findings suggested that to adapt to the variable habitats in montane forest ecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulating the absorption and composition of elements in roots

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic and terrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at wwwmdpicomxxxs1 Fig-ure S1 The mean stoichiometric homeostasis (H) values for N and P elements and their element ratios in leaf stem and root of different species Table S1 The difference of stoichiometric traits of C N and P concentrations and their element ratios in different organs of C gracilis between epi-

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic andterrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at httpswwwmdpicom1999-490712116s1 Figure S1 The mean stoichiometric homeostasis (H) values for N and Pelements and their element ratios in leaf stem and root of different species Table S1 The differenceof stoichiometric traits of C N and P concentrations and their element ratios in different organs of C

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Figure 1 The pictures of investigated species (AB) epiphytic and terrestrial individuals of Cautleya gracilis (CD) epiphytic and terrestrial individuals of Elatostema monandrum (EF) epi-phytic and terrestrial individuals of Briggsia longifolia respectively

22 Field Sampling and Greenhouse Culture In the field sampling experiment we used the natural nutrient gradients of the forest

canopy and ground soil stands to carry out nutrient additions Many factors such as light substrate water and nutrient content jointly affect the content of chemical elements in plants in the field [3234] so the relationship between a plantrsquos elemental content and its growth substrate cannot be accurately estimated by field sampling alone Therefore to accurately evaluate the stoichiometric stability of facultative epiphytes we also per-formed a greenhouse cultivation experiment in addition to the field experiment

Field sampling was done during the rainy season (August 2018) Four collection line transects were set along four evenly divided directions in the core area of montane moist evergreen broad-leaved forest in the Ailao Mountains and 3ndash5 sampling points were selected per line transect We defined individuals that grow on the trunk as epiphytic individuals and individuals that grow on the ground as terrestrial individuals The healthy mature terrestrial and epiphytic individuals of three dominant facultative epi-phytes (C gracilis E monandrum and B longifolia) were separately collected from each sampling point for which terrestrial and epiphytic individuals were collected from the forests soil and c 15 m of their host trees respectively In total 34 samples (16 samples from epiphytic individuals and 18 samples from terrestrial individuals) were collected

Figure 1 The pictures of investigated species (AB) epiphytic and terrestrial individuals of Cautleya gracilis (CD)epiphytic and terrestrial individuals of Elatostema monandrum (EF) epiphytic and terrestrial individuals of Briggsia longifoliarespectively

22 Field Sampling and Greenhouse Culture

In the field sampling experiment we used the natural nutrient gradients of the forestcanopy and ground soil stands to carry out nutrient additions Many factors such as lightsubstrate water and nutrient content jointly affect the content of chemical elements inplants in the field [3234] so the relationship between a plantrsquos elemental content and itsgrowth substrate cannot be accurately estimated by field sampling alone Therefore toaccurately evaluate the stoichiometric stability of facultative epiphytes we also performeda greenhouse cultivation experiment in addition to the field experiment

Field sampling was done during the rainy season (August 2018) Four collectionline transects were set along four evenly divided directions in the core area of montanemoist evergreen broad-leaved forest in the Ailao Mountains and 3ndash5 sampling points wereselected per line transect We defined individuals that grow on the trunk as epiphyticindividuals and individuals that grow on the ground as terrestrial individuals Thehealthy mature terrestrial and epiphytic individuals of three dominant facultative epiphytes(C gracilis E monandrum and B longifolia) were separately collected from each sampling

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point for which terrestrial and epiphytic individuals were collected from the forests soiland c 15 m of their host trees respectively In total 34 samples (16 samples from epiphyticindividuals and 18 samples from terrestrial individuals) were collected for C gracilis 30samples (18 samples from epiphytic individuals and 12 samples from terrestrial individuals)for E monandrum and 38 samples (20 samples from epiphytic individuals and 18 samplesfrom terrestrial individuals) for B longifolia We collected whole plant individuals whoseroots stems and leaves were then separated and stored as well the soil at their growthpositions of the target species for use a growing substrate for these plants These plantsamples were first washed with tap water then washed with distilled water and placed intoenvelopes and oven-dried to a constant weight (48 h) at 65 C then the dried samples wereground to powder The soil samples were dried ground and passed through a 60-meshsieve before their analysis

The cultivation experiment was carried out in a greenhouse from April to August2018 In this experiment the N and P fertilizers used were respectively NH4NO3 andNaH2PO4 their fertilization gradients are shown in Table 1 which consisted of ninetreatments in total with three replicates per treatment There were five N levels (05 1 24 and 6 mmol Lminus1) under a moderate P gradient (025 mmol Lminus1) and five P gradients(00625 0125 025 05 and 075 mmol Lminus1) under a moderate N gradient (2 mmol Lminus1) Itis difficult to collect and cultivate seedlings of C gracilis thus only the other two specieswere used for the cultivation experiment Healthy epiphytic seedlings (E monandrumand B longifolia) were collected from the montane moist evergreen broad-leaved forestin the Ailao Mountains These seedlings were pre-incubated in tap water for two weekshence the nutrient content of different individuals was made relatively equal Sterilizedvermiculite was used as growth substrate with an equal volume of vermiculite (100 mL)poured into each cultivated pot three seedlings per pot Fertilizer was added in the form ofnutrient solutions 10-mL nutrient solutions were added to the culture pots every two daysIn August 2018 the leaf stem and root of each individual were collected separately andfirst washed with tap water then washed with distilled water and placed into envelopesand oven-dried to a constant weight (48 h) at 65 C These dried samples were placed in amortar containing liquid nitrogen and powdered using a pestle each powder sample wasplaced in a small Ziplock bag Because both stems and roots did not reach the minimummass required for element analysis only the element concentrations in leaf were measured

Table 1 The N and P concentrations and ratios in nutrient solutions used in treatments of greenhousecultivation experiment

Treatment N (mmol Lminus1) P (mmol Lminus1) N P

N1P3 05 025 2N2P3 1 025 4N3P3 2 025 8N4P3 4 025 16N5P3 6 025 24P1N3 2 00625 32P2N3 2 0125 16P4N3 2 05 4P5N3 2 075 27

Five N levels under moderate P level (P3) five P levels under moderate N level (N3)

23 Laboratory Analysis

The total C and N concentrations of all plant samples in the cultivation and fieldexperiments and soil samples in the field experiment were measured with a carbon andnitrogen element analyzer (Vario MAX CN Elemental Analyzer Elementar LangenselboldGermany) about 01 g dried powder of each plant and soil sample was used for themeasurement of C and N concentrations The P concentration of plant and soil sampleswere measured by an inductively coupled plasma atomic emission spectrometer (iCAP6300Thermo Fisher Scientific Waltham MA USA) after digested with HNO3-HClO4 For the

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determination of P concentrations of plant and soil samples 048ndash052 g and 01ndash012 gdried powder was needed respectively Total C N and P concentrations of both plant andhumus soil samples were calculated on per dry weight basis

24 Data Analysis and Statistics

The stoichiometric homeostasis index (H) of different organs of each plant species wascalculated according to the ecological stoichiometric homeostasis model [17]

y = cx1H (1)

where x refers to the N or P content or the ratio of NP in the growing substrate y refers tothe content of N or P or the NP ratio in a given plant organ c is a constant In this model avalue of H gt 1 may be interpreted to mean that the plant has the ability to regulate elementalcomposition [17] Persson et al [33] classified organisms into four types according to therange of their H values as follows H gt 4 strict homeostasis 2 lt H lt 4 weakly homeostasis34 lt H lt 2 weakly plastic H lt 34 plastic

In the field experiment the differences in the C N and P elements and their stoi-chiometric ratios of facultative epiphytes between epiphytic and terrestrial habitats wereanalyzed by independent t-test and two-way ANOVA The H for N P and NP of leavesstems and roots of three species were estimated through regression analysis Variation ofH across different species organs and elements in the field experiment was analyzed byone-way ANOVA and two-way ANOVA for which statistical significance was set at p =005 Data normality and homogeneity were checked through Shapiro-Wilk and Levenetests for the data not met the assumptions Kruskal-Wallis tests were used

In the cultivation experiment the H for N P and NP of leaves were estimated throughregression analysis Variation of N P and NP in leaves of two species across differentfertilization levels were analyzed by two-way ANOVA for which statistical significancewas set at p = 005 All statistical analyses were done using IBM SPSS Statistics 190 (SPSSInc Chicago IL USA)

3 Results31 Stoichiometric Patterns of C N P Concentrations and Their Ratios in Different Organs inTwo Habitats in the Field

The C concentration and CN were only affected by species the N and P concentrationsand the CP and NP ratios in leaves were significantly affected by species and habitatwhile the CP was also affected by the interaction between species (Table 2) The C and Nconcentration and the CN CP NP ratios in stems were significantly affected by specieswhile C and CN were also affected by habitat the P concentration was only affected byhabitat For roots the C and N concentration were significantly affected by habitat andspecies the CN and NP ratios were significantly affected by species and the interactionbetween habitat and species the P and CP was only affected by species

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Table 2 Effects of species habitats and their interactions on stoichiometric traits of C N and P indifferent organs of facultative epiphytes in field experiment

Organ VariableHabitat Species Habitat times Species

F df F df F df

Leaf C 367 1 93 63420 2 93 154 2 93N 456 1 93 9122 2 93 129 2 93P 1083 1 89 4399 2 89 301 2 89

CN 056 1 93 17012 2 93 110 2 93CP 773 1 89 9448 2 89 370 2 89NP 399 1 89 453 2 89 089 2 89

Stem C 706 1 57 20677 1 57 203 1 57N 143 1 57 9392 1 57 070 1 57P 1138 1 44 284 1 44 107 1 44

CN 440 1 57 8456 1 57 082 1 57CP 374 1 44 892 1 44 002 1 44NP 324 1 44 1039 1 44 225 1 44

Root C 2339 1 90 2188 2 90 193 2 90N 469 1 90 3430 2 90 075 2 90P 338 1 65 1360 2 65 081 2 65

CN 009 1 90 6297 2 90 373 2 90CP 010 1 65 903 2 65 072 2 65NP 000 1 65 434 2 65 320 2 65

p lt 005 p lt 001 F indicates F value df denotes degree of freedom

For C gracilis the difference in the leaf N concentration and P concentration of stemand root between the two habitats was significant (p lt 005) the P concentrations of leafand the rootrsquos C and N concentrations in the epiphytic habitat significantly exceeded thosein terrestrial habitat (p lt 001) while the CN and CP in leaf between the two habitatsshowed a reverse trend (Table S1) For E monandrum the C and P concentration in leaf andthe C and N concentration and the CN ratio in root in the epiphytic habitat were higherthan those in terrestrial habitat (Table S2) For B longifolia the C N and P concentrations inleaf and root in the epiphytic habitat were higher than those in terrestrial habitat but viceversa for the CN and CP in leaf (Table S3) At the species level the plasticity of elementcomposition in epiphytic individuals of the three species were relatively higher than that ofterrestrial individuals expect C in root and P in stem of C gracilis C and N in leaf and rootof E monandrum and C and N in leaf and root CN in leaf of B longifolia (Tables S1ndashS3)

32 Variation of Stoichiometric Homeostasis among Different Species Organs and Elements inthe Field

For leaves the HN of C gracilis was weaker than either that of E monandrum orB longifolia while the latter was characterized by the strongest N homeostasis (Figure 2Table S4) The HP of species was ranked in the order of E monandrum gt C gracilis gt Blongifolia In terms of HNP it was strongest in C gracilis followed by E monandrum beingweakest in B longifolia For stems with the exception of NP E monandrum had H valuesfor N and P that were each stronger than those characterizing C gracilis For roots exceptfor HN the H values for P and NP descended among species in the order of B longifoliagt C gracilis gt E monandrum Whereas the corresponding order for HN was B longifolia gtE monandrum gt C gracilis For the mean values of H the difference of leaves and rootsamong three species and stems between two species were all not significant (Figure S1)

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Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stemand (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrumB longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species allstronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stemsand roots the mean values for HN and HP of the three species were all decreased in theorder of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems thehomeostasis for NP was stronger than the P element but weaker than the N element Inroots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gtHN gt HP)

Forests 2021 12 x FOR PEER REVIEW 8 of 15

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratiosin leaves stems and roots of three species Bars are the mean values with standard error Capital andlowercase letters indicate significant differences among organs and element types respectively

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment

In the cultivation experiment the concentration of N and P in leaves were significantlyaffected by species and treatment levels NP was significantly affected by the treatment level

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while the interaction between species and treatment level had no significant effects on theN and P concentration and their elemental ratio (Table 3) The N and P concentrations ofleaves were both increased as higher N and P concentrations were applied in solution For thestoichiometric homeostasis values of different elements of leaves in the two species all valueswere greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the Nelement was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their interactionon N and P concentrations and NP ratios in leaves of two species in the cultivation experiment

VariableSpecies Treatment Species times Level

F df p F df p F df p

N 22611 1 000 417 8 003 132 8 022P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098The numerator df are given in the table the denominator df is 8

Forests 2021 12 x FOR PEER REVIEW 9 of 15

treatment level while the interaction between species and treatment level had no signif-icant effects on the N and P concentration and their elemental ratio (Table 3) The N and P concentrations of leaves were both increased as higher N and P concentrations were ap-plied in solution For the stoichiometric homeostasis values of different elements of leaves in the two species all values were greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the N element was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their in-teraction on N and P concentrations and NP ratios in leaves of two species in the cultivation ex-periment

Variable Species Treatment Species times Level

F df p F df p F df p N 22611 1 000 417 8 003 132 8 022 P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098 The numerator df are given in the table the denominator df is 8

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N P and NP in the greenhouse cultivation experiment (a b c) H values for leaf N P and N P of Briggsia longifolia (d e f) H values for leaf N P and N P of Elatostema monandrum respectively

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N Pand NP in the greenhouse cultivation experiment (andashc) H values for leaf N P and N P of Briggsialongifolia (dndashf) H values for leaf N P and N P of Elatostema monandrum respectively

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4 Discussion41 Differences in Nutrient Use Strategy between Epiphytic and Terrestrial Individuals ofFacultative Epiphytes

In our field experiment the concentrations of N and P in epiphytic individuals ofthree species in the natural forest were higher than those in terrestrial individuals but CNCP and NP ratios in the former were lower than the latter (Tables S1ndashS3) This agreedwith the results from fern in our study area and bromeliads in humid montane forest andlowland forests elsewhere [134748] CN and CP ratios reflect the carbon fixation per unitnutrient the higher N and P concentration and lower CN and CP values are associatedwith luxury consumption of plants [164950] Accordingly in our study the use of N andP by epiphytic individuals were more luxurious than terrestrial individuals which agreeswith the result that terrestrial individuals of fern are more efficient in nutrient use thanepiphytic individuals [13] The plasticity in nutrient use strategy of facultative epiphytes isconsidered to be an adaptive mechanism to different environmental conditions in orderto exploit the available resources efficiently [1213] Our results indicated that facultativeepiphytes had more flexible plasticity in nutrient use strategies to adapt habitat shifts

Winkler and Zotz [51] found that epiphytes possessed lower nutrient use efficiencythan that of terrestrial plants Epiphytes are generally considered to survive in the nutrientintermittent epiphytic habitats through luxurious consumption of nutrients [12] Theyusually absorb large amounts of nutrients and store them in vacuoles when the nutrient isabundant [142949] Accordingly the lower N and P use of epiphytic individuals may berelated to the luxury uptake of N and P in epiphytic habitats Furthermore we found thatvariation in the elemental composition of epiphytic individuals exceeded that of terrestrialindividuals most likely because epiphytic habitats are more heterogeneous [552] and theelemental composition of plants is influenced by other environmental factors (eg wateravailability light intensity) besides soil alone [3849]

42 Effects of Nutrient Limitation on the Distribution of Three Facultative Epiphytes

The foliar NP ratio can be used as an indicator of the type of nutrient limitationon plant growth the lower NP ratios indicate the plants were limited by N [244953]whereas the higher NP ratios indicates P limitations were present [2954] Epiphytes areusually restricted by P [55] but in our research nutrient restriction types differ amongthe three species The NP ratio of terrestrial and epiphytic individuals of E monandrumand epiphytic individuals of B longifolia was lower than 12 (p-values = 0214 0526 0225respectively) (Tables S2 and S3) suggesting the growth of which was restricted by Navailability [29] Since the NP ratio in terrestrial and epiphytic individuals of C gracilisand terrestrial individuals of B longifolia was higher than 12 (p-values = 0004 0227 0009respectively) yet still lower than 16 (p-values = 0 005 0 0006 respectively) (Tables S1 andS3) we interpreted this to mean that the growth of which were limited by N or co-limited byN and P [28] Our results were consistent with the finding that facultative epiphytes on datepalms (Phoenix dactylifera) in southeastern Spain were subject to N-limited conditions [14]Furthermore in our research the lower NP ratio in epiphytic than terrestrial individualsimplied that N-restriction in the epiphytic habitat was more pronounced The higher Pconcentration in epiphytic individuals probably increases the demands for N thus makingthem more pronounced Furthermore the higher P concentration in plants can alleviate thedamage caused by drought stress [49] In this study the higher P concentration in epiphyticindividuals may reflect their adaptation to epiphytic habitats where water is intermittent

The factors that drive the distribution of facultative epiphytes are not clear butprevious results suggested that environmental factors can affect the distribution of epi-phytes [1446] For the three species in our research most individuals of E monandrum andB longifolia grow on the bark with mass mat or thick humus while the majority of C gracilisgrow on the forest floor For E monandrum and B longifolia the N-restriction in epiphytichabitats was stronger than terrestrial habitats therefore they may gradually shift to forestfloor to alleviate N limitation For C gracilis the type of nutrient limitation in terrestrial and

Forests 2021 12 16 11 of 15

epiphytic habitats was same this indicated that the nutrient status in epiphytic habitatscan satisfy its requirement Previous research indicated that the interspecific competition interrestrial habitats was more intense than that in epiphytic habitats [7] Therefore C gracilismay shift to epiphytic habitats to escape interspecific competition

43 Differences in Stoichiometric Homeostasis among Species Organs and Elements ofFacultative Epiphytes

Traditional theory predicts that the stoichiometric homeostasis of autotroph was rela-tively weak [1756] However mounting data suggest that plants have a strong regulatorycapacity for their element composition [202237] Our results of the field and greenhousecultivation experiment indicated that the values of HN HP and HNP of leaves were allgt4 (p = 0002 0004 respectively) (Figure 4 Table S4) both showed strong homeostasiswhich did not support our second hypothesis that the homeostasis of facultative epiphytesis weak These results for facultative epiphytes in our study are also at odds with previousresearch concluded that a weak homeostasis of plants implied higher ecological adapt-ability and better suitability to a more variable environment [33] This discrepancy maybe because that conclusion about relationship between stoichiometric homeostasis andecological adaptability of plants was almost based on studies of plant leaves [3349] Theelemental composition of leaves is insensitive to nutrient changes in the soil instead rootsare more valid indicators of nutrient change in the environment [203857ndash59] Therefore aleafrsquos stoichiometric homeostasis cannot accurately reflect the ecological adaptability ofplants [3860] In our work here although leaves did have strong stoichiometric home-ostasis it was rather weak for roots (Table S4) The main function of roots is to absorbmineral nutrients and water from the soil [33] their plastic nutrient absorption is helpfulfor facultative epiphytes to efficiently exploit the available resources in different habitatsThis result indicated that facultative epiphytes could adapt to variable environment bychanging the elemental composition of their roots

Moreover the difference in stoichiometric homeostasis among the three species in ourfield study was not significant and the stoichiometric homeostasis mainly affected by or-gans (Table S5) likely because all three share a similar species richness in the sampling areaand their ecological strategy and adaptability to the environment were comparable [22]This was also confirmed by the consistent response of the two species to nutrient additionin the greenhouse cultivation experiments in our study (Table 3)

Different organs of the same species exhibit disparate stoichiometric characteristicsto better adapt to the environment [162033] We found that stoichiometric homeostasiswas stronger in aboveground than belowground of the three facultative epiphytes Rootswere more sensitive to changes of elements concentrations in environmental resourceswhich agrees with findings reported for terrestrial grass and woody plants [21223138]However unlike the related studies on terrestrial woody plants [38] the stems of C gracilisand E monandrum show strong stoichiometric homeostasis (Table S4) This may be relatedto the insensitivity of some key elements in stems of facultative epiphyte to changes insoil nutrient concentrations The stems of woody and herbaceous plants take differentfunctions the stems of woody plants mainly play transportation and supporting rolewhile the stems of herbaceous plants have less structural C than woody plants yet bearthe functions of photosynthesis transportation and the supporting action [20] Thereforethe stems of herbaceous plants need relatively stable supply of elements to maintain theirphysiological functions

The strength of homeostasis is also related to the elemental concentration [61] Previ-ous research for terrestrial plants indicated that the homeostasis of macro-elements washigher than trace elements [3461] In our study the value of stoichiometric homeostasisof N and P in the three facultative epiphyte species showed the order of HN gt HP This isconsistent with results from vertebrates and trees with greater control over high-demandelements [34ndash36] Therefore the facultative epiphytic plants may also have greater con-trol over macro-elements highly demanded for physiology functioning However ourresearch only focused on the stoichiometric homeostasis of N and P elements in facultative

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epiphytes the stoichiometric homeostasis of trace elements remains unknown Thereforewhether the stoichiometric homeostasis of macro-elements in facultative epiphytes is higherthan trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumulationof N within a given plant tissue is usually accompanied by an increase in its P content thehomeostasis of NP is stronger than N and P elements [1762] However in our study thestoichiometric homeostasis value of NP in leaf and root were all higher than that of P butlower than that of N (Figure 3) Based on the theory that limiting elements should havestrong homeostasis [313463] we speculate that facultative epiphytes may be restricted byN availability These plants therefore would take strong regulation of N to cope with theunpredictable fluctuations of N elements in their habitat

5 Conclusions

This study systematically elucidates the nutrient use and ecological adaptation strate-gies of facultative epiphytes from the perspective of stoichiometry using field investigationand greenhouse cultivation experiments Our study showed that epiphytic individualsof facultative epiphytes survive intermittent habitat via luxury consumption of nutrientswhile terrestrial individuals use more conservative nutrients (Figure 5) The nutrient usestrategies of facultative epiphytes were flexible which enabled facultative epiphytes toexploit bark and soil interchangeably Facultative epiphytes had strong control over N andP concentrations and NP ratio of leaves and stems while roots had weaker stoichiometricstability These findings suggested that to adapt to the variable habitats in montane forestecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulatingthe absorption and composition of elements in roots

|

Forests 2021 12 x FOR PEER REVIEW 12 of 15

trol over macro-elements highly demanded for physiology functioning However our research only focused on the stoichiometric homeostasis of N and P elements in faculta-tive epiphytes the stoichiometric homeostasis of trace elements remains unknown Therefore whether the stoichiometric homeostasis of macro-elements in facultative epi-phytes is higher than trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumu-lation of N within a given plant tissue is usually accompanied by an increase in its P content the homeostasis of NP is stronger than N and P elements [1762] However in our study the stoichiometric homeostasis value of NP in leaf and root were all higher than that of P but lower than that of N (Figure 3) Based on the theory that limiting ele-ments should have strong homeostasis [313463] we speculate that facultative epiphytes may be restricted by N availability These plants therefore would take strong regulation of N to cope with the unpredictable fluctuations of N elements in their habitat

5 Conclusions This study systematically elucidates the nutrient use and ecological adaptation

strategies of facultative epiphytes from the perspective of stoichiometry using field in-vestigation and greenhouse cultivation experiments Our study showed that epiphytic individuals of facultative epiphytes survive intermittent habitat via luxury consumption of nutrients while terrestrial individuals use more conservative nutrients (Figure 5) The nutrient use strategies of facultative epiphytes were flexible which enabled facultative epiphytes to exploit bark and soil interchangeably Facultative epiphytes had strong control over N and P concentrations and NP ratio of leaves and stems while roots had weaker stoichiometric stability These findings suggested that to adapt to the variable habitats in montane forest ecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulating the absorption and composition of elements in roots

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic and terrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at wwwmdpicomxxxs1 Fig-ure S1 The mean stoichiometric homeostasis (H) values for N and P elements and their element ratios in leaf stem and root of different species Table S1 The difference of stoichiometric traits of C N and P concentrations and their element ratios in different organs of C gracilis between epi-

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic andterrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at httpswwwmdpicom1999-490712116s1 Figure S1 The mean stoichiometric homeostasis (H) values for N and Pelements and their element ratios in leaf stem and root of different species Table S1 The differenceof stoichiometric traits of C N and P concentrations and their element ratios in different organs of C

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point for which terrestrial and epiphytic individuals were collected from the forests soiland c 15 m of their host trees respectively In total 34 samples (16 samples from epiphyticindividuals and 18 samples from terrestrial individuals) were collected for C gracilis 30samples (18 samples from epiphytic individuals and 12 samples from terrestrial individuals)for E monandrum and 38 samples (20 samples from epiphytic individuals and 18 samplesfrom terrestrial individuals) for B longifolia We collected whole plant individuals whoseroots stems and leaves were then separated and stored as well the soil at their growthpositions of the target species for use a growing substrate for these plants These plantsamples were first washed with tap water then washed with distilled water and placed intoenvelopes and oven-dried to a constant weight (48 h) at 65 C then the dried samples wereground to powder The soil samples were dried ground and passed through a 60-meshsieve before their analysis

The cultivation experiment was carried out in a greenhouse from April to August2018 In this experiment the N and P fertilizers used were respectively NH4NO3 andNaH2PO4 their fertilization gradients are shown in Table 1 which consisted of ninetreatments in total with three replicates per treatment There were five N levels (05 1 24 and 6 mmol Lminus1) under a moderate P gradient (025 mmol Lminus1) and five P gradients(00625 0125 025 05 and 075 mmol Lminus1) under a moderate N gradient (2 mmol Lminus1) Itis difficult to collect and cultivate seedlings of C gracilis thus only the other two specieswere used for the cultivation experiment Healthy epiphytic seedlings (E monandrumand B longifolia) were collected from the montane moist evergreen broad-leaved forestin the Ailao Mountains These seedlings were pre-incubated in tap water for two weekshence the nutrient content of different individuals was made relatively equal Sterilizedvermiculite was used as growth substrate with an equal volume of vermiculite (100 mL)poured into each cultivated pot three seedlings per pot Fertilizer was added in the form ofnutrient solutions 10-mL nutrient solutions were added to the culture pots every two daysIn August 2018 the leaf stem and root of each individual were collected separately andfirst washed with tap water then washed with distilled water and placed into envelopesand oven-dried to a constant weight (48 h) at 65 C These dried samples were placed in amortar containing liquid nitrogen and powdered using a pestle each powder sample wasplaced in a small Ziplock bag Because both stems and roots did not reach the minimummass required for element analysis only the element concentrations in leaf were measured

Table 1 The N and P concentrations and ratios in nutrient solutions used in treatments of greenhousecultivation experiment

Treatment N (mmol Lminus1) P (mmol Lminus1) N P

N1P3 05 025 2N2P3 1 025 4N3P3 2 025 8N4P3 4 025 16N5P3 6 025 24P1N3 2 00625 32P2N3 2 0125 16P4N3 2 05 4P5N3 2 075 27

Five N levels under moderate P level (P3) five P levels under moderate N level (N3)

23 Laboratory Analysis

The total C and N concentrations of all plant samples in the cultivation and fieldexperiments and soil samples in the field experiment were measured with a carbon andnitrogen element analyzer (Vario MAX CN Elemental Analyzer Elementar LangenselboldGermany) about 01 g dried powder of each plant and soil sample was used for themeasurement of C and N concentrations The P concentration of plant and soil sampleswere measured by an inductively coupled plasma atomic emission spectrometer (iCAP6300Thermo Fisher Scientific Waltham MA USA) after digested with HNO3-HClO4 For the

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determination of P concentrations of plant and soil samples 048ndash052 g and 01ndash012 gdried powder was needed respectively Total C N and P concentrations of both plant andhumus soil samples were calculated on per dry weight basis

24 Data Analysis and Statistics

The stoichiometric homeostasis index (H) of different organs of each plant species wascalculated according to the ecological stoichiometric homeostasis model [17]

y = cx1H (1)

where x refers to the N or P content or the ratio of NP in the growing substrate y refers tothe content of N or P or the NP ratio in a given plant organ c is a constant In this model avalue of H gt 1 may be interpreted to mean that the plant has the ability to regulate elementalcomposition [17] Persson et al [33] classified organisms into four types according to therange of their H values as follows H gt 4 strict homeostasis 2 lt H lt 4 weakly homeostasis34 lt H lt 2 weakly plastic H lt 34 plastic

In the field experiment the differences in the C N and P elements and their stoi-chiometric ratios of facultative epiphytes between epiphytic and terrestrial habitats wereanalyzed by independent t-test and two-way ANOVA The H for N P and NP of leavesstems and roots of three species were estimated through regression analysis Variation ofH across different species organs and elements in the field experiment was analyzed byone-way ANOVA and two-way ANOVA for which statistical significance was set at p =005 Data normality and homogeneity were checked through Shapiro-Wilk and Levenetests for the data not met the assumptions Kruskal-Wallis tests were used

In the cultivation experiment the H for N P and NP of leaves were estimated throughregression analysis Variation of N P and NP in leaves of two species across differentfertilization levels were analyzed by two-way ANOVA for which statistical significancewas set at p = 005 All statistical analyses were done using IBM SPSS Statistics 190 (SPSSInc Chicago IL USA)

3 Results31 Stoichiometric Patterns of C N P Concentrations and Their Ratios in Different Organs inTwo Habitats in the Field

The C concentration and CN were only affected by species the N and P concentrationsand the CP and NP ratios in leaves were significantly affected by species and habitatwhile the CP was also affected by the interaction between species (Table 2) The C and Nconcentration and the CN CP NP ratios in stems were significantly affected by specieswhile C and CN were also affected by habitat the P concentration was only affected byhabitat For roots the C and N concentration were significantly affected by habitat andspecies the CN and NP ratios were significantly affected by species and the interactionbetween habitat and species the P and CP was only affected by species

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Table 2 Effects of species habitats and their interactions on stoichiometric traits of C N and P indifferent organs of facultative epiphytes in field experiment

Organ VariableHabitat Species Habitat times Species

F df F df F df

Leaf C 367 1 93 63420 2 93 154 2 93N 456 1 93 9122 2 93 129 2 93P 1083 1 89 4399 2 89 301 2 89

CN 056 1 93 17012 2 93 110 2 93CP 773 1 89 9448 2 89 370 2 89NP 399 1 89 453 2 89 089 2 89

Stem C 706 1 57 20677 1 57 203 1 57N 143 1 57 9392 1 57 070 1 57P 1138 1 44 284 1 44 107 1 44

CN 440 1 57 8456 1 57 082 1 57CP 374 1 44 892 1 44 002 1 44NP 324 1 44 1039 1 44 225 1 44

Root C 2339 1 90 2188 2 90 193 2 90N 469 1 90 3430 2 90 075 2 90P 338 1 65 1360 2 65 081 2 65

CN 009 1 90 6297 2 90 373 2 90CP 010 1 65 903 2 65 072 2 65NP 000 1 65 434 2 65 320 2 65

p lt 005 p lt 001 F indicates F value df denotes degree of freedom

For C gracilis the difference in the leaf N concentration and P concentration of stemand root between the two habitats was significant (p lt 005) the P concentrations of leafand the rootrsquos C and N concentrations in the epiphytic habitat significantly exceeded thosein terrestrial habitat (p lt 001) while the CN and CP in leaf between the two habitatsshowed a reverse trend (Table S1) For E monandrum the C and P concentration in leaf andthe C and N concentration and the CN ratio in root in the epiphytic habitat were higherthan those in terrestrial habitat (Table S2) For B longifolia the C N and P concentrations inleaf and root in the epiphytic habitat were higher than those in terrestrial habitat but viceversa for the CN and CP in leaf (Table S3) At the species level the plasticity of elementcomposition in epiphytic individuals of the three species were relatively higher than that ofterrestrial individuals expect C in root and P in stem of C gracilis C and N in leaf and rootof E monandrum and C and N in leaf and root CN in leaf of B longifolia (Tables S1ndashS3)

32 Variation of Stoichiometric Homeostasis among Different Species Organs and Elements inthe Field

For leaves the HN of C gracilis was weaker than either that of E monandrum orB longifolia while the latter was characterized by the strongest N homeostasis (Figure 2Table S4) The HP of species was ranked in the order of E monandrum gt C gracilis gt Blongifolia In terms of HNP it was strongest in C gracilis followed by E monandrum beingweakest in B longifolia For stems with the exception of NP E monandrum had H valuesfor N and P that were each stronger than those characterizing C gracilis For roots exceptfor HN the H values for P and NP descended among species in the order of B longifoliagt C gracilis gt E monandrum Whereas the corresponding order for HN was B longifolia gtE monandrum gt C gracilis For the mean values of H the difference of leaves and rootsamong three species and stems between two species were all not significant (Figure S1)

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Forests 2021 12 x FOR PEER REVIEW 8 of 15

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stemand (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrumB longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species allstronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stemsand roots the mean values for HN and HP of the three species were all decreased in theorder of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems thehomeostasis for NP was stronger than the P element but weaker than the N element Inroots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gtHN gt HP)

Forests 2021 12 x FOR PEER REVIEW 8 of 15

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratiosin leaves stems and roots of three species Bars are the mean values with standard error Capital andlowercase letters indicate significant differences among organs and element types respectively

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment

In the cultivation experiment the concentration of N and P in leaves were significantlyaffected by species and treatment levels NP was significantly affected by the treatment level

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while the interaction between species and treatment level had no significant effects on theN and P concentration and their elemental ratio (Table 3) The N and P concentrations ofleaves were both increased as higher N and P concentrations were applied in solution For thestoichiometric homeostasis values of different elements of leaves in the two species all valueswere greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the Nelement was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their interactionon N and P concentrations and NP ratios in leaves of two species in the cultivation experiment

VariableSpecies Treatment Species times Level

F df p F df p F df p

N 22611 1 000 417 8 003 132 8 022P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098The numerator df are given in the table the denominator df is 8

Forests 2021 12 x FOR PEER REVIEW 9 of 15

treatment level while the interaction between species and treatment level had no signif-icant effects on the N and P concentration and their elemental ratio (Table 3) The N and P concentrations of leaves were both increased as higher N and P concentrations were ap-plied in solution For the stoichiometric homeostasis values of different elements of leaves in the two species all values were greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the N element was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their in-teraction on N and P concentrations and NP ratios in leaves of two species in the cultivation ex-periment

Variable Species Treatment Species times Level

F df p F df p F df p N 22611 1 000 417 8 003 132 8 022 P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098 The numerator df are given in the table the denominator df is 8

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N P and NP in the greenhouse cultivation experiment (a b c) H values for leaf N P and N P of Briggsia longifolia (d e f) H values for leaf N P and N P of Elatostema monandrum respectively

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N Pand NP in the greenhouse cultivation experiment (andashc) H values for leaf N P and N P of Briggsialongifolia (dndashf) H values for leaf N P and N P of Elatostema monandrum respectively

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4 Discussion41 Differences in Nutrient Use Strategy between Epiphytic and Terrestrial Individuals ofFacultative Epiphytes

In our field experiment the concentrations of N and P in epiphytic individuals ofthree species in the natural forest were higher than those in terrestrial individuals but CNCP and NP ratios in the former were lower than the latter (Tables S1ndashS3) This agreedwith the results from fern in our study area and bromeliads in humid montane forest andlowland forests elsewhere [134748] CN and CP ratios reflect the carbon fixation per unitnutrient the higher N and P concentration and lower CN and CP values are associatedwith luxury consumption of plants [164950] Accordingly in our study the use of N andP by epiphytic individuals were more luxurious than terrestrial individuals which agreeswith the result that terrestrial individuals of fern are more efficient in nutrient use thanepiphytic individuals [13] The plasticity in nutrient use strategy of facultative epiphytes isconsidered to be an adaptive mechanism to different environmental conditions in orderto exploit the available resources efficiently [1213] Our results indicated that facultativeepiphytes had more flexible plasticity in nutrient use strategies to adapt habitat shifts

Winkler and Zotz [51] found that epiphytes possessed lower nutrient use efficiencythan that of terrestrial plants Epiphytes are generally considered to survive in the nutrientintermittent epiphytic habitats through luxurious consumption of nutrients [12] Theyusually absorb large amounts of nutrients and store them in vacuoles when the nutrient isabundant [142949] Accordingly the lower N and P use of epiphytic individuals may berelated to the luxury uptake of N and P in epiphytic habitats Furthermore we found thatvariation in the elemental composition of epiphytic individuals exceeded that of terrestrialindividuals most likely because epiphytic habitats are more heterogeneous [552] and theelemental composition of plants is influenced by other environmental factors (eg wateravailability light intensity) besides soil alone [3849]

42 Effects of Nutrient Limitation on the Distribution of Three Facultative Epiphytes

The foliar NP ratio can be used as an indicator of the type of nutrient limitationon plant growth the lower NP ratios indicate the plants were limited by N [244953]whereas the higher NP ratios indicates P limitations were present [2954] Epiphytes areusually restricted by P [55] but in our research nutrient restriction types differ amongthe three species The NP ratio of terrestrial and epiphytic individuals of E monandrumand epiphytic individuals of B longifolia was lower than 12 (p-values = 0214 0526 0225respectively) (Tables S2 and S3) suggesting the growth of which was restricted by Navailability [29] Since the NP ratio in terrestrial and epiphytic individuals of C gracilisand terrestrial individuals of B longifolia was higher than 12 (p-values = 0004 0227 0009respectively) yet still lower than 16 (p-values = 0 005 0 0006 respectively) (Tables S1 andS3) we interpreted this to mean that the growth of which were limited by N or co-limited byN and P [28] Our results were consistent with the finding that facultative epiphytes on datepalms (Phoenix dactylifera) in southeastern Spain were subject to N-limited conditions [14]Furthermore in our research the lower NP ratio in epiphytic than terrestrial individualsimplied that N-restriction in the epiphytic habitat was more pronounced The higher Pconcentration in epiphytic individuals probably increases the demands for N thus makingthem more pronounced Furthermore the higher P concentration in plants can alleviate thedamage caused by drought stress [49] In this study the higher P concentration in epiphyticindividuals may reflect their adaptation to epiphytic habitats where water is intermittent

The factors that drive the distribution of facultative epiphytes are not clear butprevious results suggested that environmental factors can affect the distribution of epi-phytes [1446] For the three species in our research most individuals of E monandrum andB longifolia grow on the bark with mass mat or thick humus while the majority of C gracilisgrow on the forest floor For E monandrum and B longifolia the N-restriction in epiphytichabitats was stronger than terrestrial habitats therefore they may gradually shift to forestfloor to alleviate N limitation For C gracilis the type of nutrient limitation in terrestrial and

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epiphytic habitats was same this indicated that the nutrient status in epiphytic habitatscan satisfy its requirement Previous research indicated that the interspecific competition interrestrial habitats was more intense than that in epiphytic habitats [7] Therefore C gracilismay shift to epiphytic habitats to escape interspecific competition

43 Differences in Stoichiometric Homeostasis among Species Organs and Elements ofFacultative Epiphytes

Traditional theory predicts that the stoichiometric homeostasis of autotroph was rela-tively weak [1756] However mounting data suggest that plants have a strong regulatorycapacity for their element composition [202237] Our results of the field and greenhousecultivation experiment indicated that the values of HN HP and HNP of leaves were allgt4 (p = 0002 0004 respectively) (Figure 4 Table S4) both showed strong homeostasiswhich did not support our second hypothesis that the homeostasis of facultative epiphytesis weak These results for facultative epiphytes in our study are also at odds with previousresearch concluded that a weak homeostasis of plants implied higher ecological adapt-ability and better suitability to a more variable environment [33] This discrepancy maybe because that conclusion about relationship between stoichiometric homeostasis andecological adaptability of plants was almost based on studies of plant leaves [3349] Theelemental composition of leaves is insensitive to nutrient changes in the soil instead rootsare more valid indicators of nutrient change in the environment [203857ndash59] Therefore aleafrsquos stoichiometric homeostasis cannot accurately reflect the ecological adaptability ofplants [3860] In our work here although leaves did have strong stoichiometric home-ostasis it was rather weak for roots (Table S4) The main function of roots is to absorbmineral nutrients and water from the soil [33] their plastic nutrient absorption is helpfulfor facultative epiphytes to efficiently exploit the available resources in different habitatsThis result indicated that facultative epiphytes could adapt to variable environment bychanging the elemental composition of their roots

Moreover the difference in stoichiometric homeostasis among the three species in ourfield study was not significant and the stoichiometric homeostasis mainly affected by or-gans (Table S5) likely because all three share a similar species richness in the sampling areaand their ecological strategy and adaptability to the environment were comparable [22]This was also confirmed by the consistent response of the two species to nutrient additionin the greenhouse cultivation experiments in our study (Table 3)

Different organs of the same species exhibit disparate stoichiometric characteristicsto better adapt to the environment [162033] We found that stoichiometric homeostasiswas stronger in aboveground than belowground of the three facultative epiphytes Rootswere more sensitive to changes of elements concentrations in environmental resourceswhich agrees with findings reported for terrestrial grass and woody plants [21223138]However unlike the related studies on terrestrial woody plants [38] the stems of C gracilisand E monandrum show strong stoichiometric homeostasis (Table S4) This may be relatedto the insensitivity of some key elements in stems of facultative epiphyte to changes insoil nutrient concentrations The stems of woody and herbaceous plants take differentfunctions the stems of woody plants mainly play transportation and supporting rolewhile the stems of herbaceous plants have less structural C than woody plants yet bearthe functions of photosynthesis transportation and the supporting action [20] Thereforethe stems of herbaceous plants need relatively stable supply of elements to maintain theirphysiological functions

The strength of homeostasis is also related to the elemental concentration [61] Previ-ous research for terrestrial plants indicated that the homeostasis of macro-elements washigher than trace elements [3461] In our study the value of stoichiometric homeostasisof N and P in the three facultative epiphyte species showed the order of HN gt HP This isconsistent with results from vertebrates and trees with greater control over high-demandelements [34ndash36] Therefore the facultative epiphytic plants may also have greater con-trol over macro-elements highly demanded for physiology functioning However ourresearch only focused on the stoichiometric homeostasis of N and P elements in facultative

Forests 2021 12 16 12 of 15

epiphytes the stoichiometric homeostasis of trace elements remains unknown Thereforewhether the stoichiometric homeostasis of macro-elements in facultative epiphytes is higherthan trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumulationof N within a given plant tissue is usually accompanied by an increase in its P content thehomeostasis of NP is stronger than N and P elements [1762] However in our study thestoichiometric homeostasis value of NP in leaf and root were all higher than that of P butlower than that of N (Figure 3) Based on the theory that limiting elements should havestrong homeostasis [313463] we speculate that facultative epiphytes may be restricted byN availability These plants therefore would take strong regulation of N to cope with theunpredictable fluctuations of N elements in their habitat

5 Conclusions

This study systematically elucidates the nutrient use and ecological adaptation strate-gies of facultative epiphytes from the perspective of stoichiometry using field investigationand greenhouse cultivation experiments Our study showed that epiphytic individualsof facultative epiphytes survive intermittent habitat via luxury consumption of nutrientswhile terrestrial individuals use more conservative nutrients (Figure 5) The nutrient usestrategies of facultative epiphytes were flexible which enabled facultative epiphytes toexploit bark and soil interchangeably Facultative epiphytes had strong control over N andP concentrations and NP ratio of leaves and stems while roots had weaker stoichiometricstability These findings suggested that to adapt to the variable habitats in montane forestecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulatingthe absorption and composition of elements in roots

|

Forests 2021 12 x FOR PEER REVIEW 12 of 15

trol over macro-elements highly demanded for physiology functioning However our research only focused on the stoichiometric homeostasis of N and P elements in faculta-tive epiphytes the stoichiometric homeostasis of trace elements remains unknown Therefore whether the stoichiometric homeostasis of macro-elements in facultative epi-phytes is higher than trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumu-lation of N within a given plant tissue is usually accompanied by an increase in its P content the homeostasis of NP is stronger than N and P elements [1762] However in our study the stoichiometric homeostasis value of NP in leaf and root were all higher than that of P but lower than that of N (Figure 3) Based on the theory that limiting ele-ments should have strong homeostasis [313463] we speculate that facultative epiphytes may be restricted by N availability These plants therefore would take strong regulation of N to cope with the unpredictable fluctuations of N elements in their habitat

5 Conclusions This study systematically elucidates the nutrient use and ecological adaptation

strategies of facultative epiphytes from the perspective of stoichiometry using field in-vestigation and greenhouse cultivation experiments Our study showed that epiphytic individuals of facultative epiphytes survive intermittent habitat via luxury consumption of nutrients while terrestrial individuals use more conservative nutrients (Figure 5) The nutrient use strategies of facultative epiphytes were flexible which enabled facultative epiphytes to exploit bark and soil interchangeably Facultative epiphytes had strong control over N and P concentrations and NP ratio of leaves and stems while roots had weaker stoichiometric stability These findings suggested that to adapt to the variable habitats in montane forest ecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulating the absorption and composition of elements in roots

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic and terrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at wwwmdpicomxxxs1 Fig-ure S1 The mean stoichiometric homeostasis (H) values for N and P elements and their element ratios in leaf stem and root of different species Table S1 The difference of stoichiometric traits of C N and P concentrations and their element ratios in different organs of C gracilis between epi-

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic andterrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at httpswwwmdpicom1999-490712116s1 Figure S1 The mean stoichiometric homeostasis (H) values for N and Pelements and their element ratios in leaf stem and root of different species Table S1 The differenceof stoichiometric traits of C N and P concentrations and their element ratios in different organs of C

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determination of P concentrations of plant and soil samples 048ndash052 g and 01ndash012 gdried powder was needed respectively Total C N and P concentrations of both plant andhumus soil samples were calculated on per dry weight basis

24 Data Analysis and Statistics

The stoichiometric homeostasis index (H) of different organs of each plant species wascalculated according to the ecological stoichiometric homeostasis model [17]

y = cx1H (1)

where x refers to the N or P content or the ratio of NP in the growing substrate y refers tothe content of N or P or the NP ratio in a given plant organ c is a constant In this model avalue of H gt 1 may be interpreted to mean that the plant has the ability to regulate elementalcomposition [17] Persson et al [33] classified organisms into four types according to therange of their H values as follows H gt 4 strict homeostasis 2 lt H lt 4 weakly homeostasis34 lt H lt 2 weakly plastic H lt 34 plastic

In the field experiment the differences in the C N and P elements and their stoi-chiometric ratios of facultative epiphytes between epiphytic and terrestrial habitats wereanalyzed by independent t-test and two-way ANOVA The H for N P and NP of leavesstems and roots of three species were estimated through regression analysis Variation ofH across different species organs and elements in the field experiment was analyzed byone-way ANOVA and two-way ANOVA for which statistical significance was set at p =005 Data normality and homogeneity were checked through Shapiro-Wilk and Levenetests for the data not met the assumptions Kruskal-Wallis tests were used

In the cultivation experiment the H for N P and NP of leaves were estimated throughregression analysis Variation of N P and NP in leaves of two species across differentfertilization levels were analyzed by two-way ANOVA for which statistical significancewas set at p = 005 All statistical analyses were done using IBM SPSS Statistics 190 (SPSSInc Chicago IL USA)

3 Results31 Stoichiometric Patterns of C N P Concentrations and Their Ratios in Different Organs inTwo Habitats in the Field

The C concentration and CN were only affected by species the N and P concentrationsand the CP and NP ratios in leaves were significantly affected by species and habitatwhile the CP was also affected by the interaction between species (Table 2) The C and Nconcentration and the CN CP NP ratios in stems were significantly affected by specieswhile C and CN were also affected by habitat the P concentration was only affected byhabitat For roots the C and N concentration were significantly affected by habitat andspecies the CN and NP ratios were significantly affected by species and the interactionbetween habitat and species the P and CP was only affected by species

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Table 2 Effects of species habitats and their interactions on stoichiometric traits of C N and P indifferent organs of facultative epiphytes in field experiment

Organ VariableHabitat Species Habitat times Species

F df F df F df

Leaf C 367 1 93 63420 2 93 154 2 93N 456 1 93 9122 2 93 129 2 93P 1083 1 89 4399 2 89 301 2 89

CN 056 1 93 17012 2 93 110 2 93CP 773 1 89 9448 2 89 370 2 89NP 399 1 89 453 2 89 089 2 89

Stem C 706 1 57 20677 1 57 203 1 57N 143 1 57 9392 1 57 070 1 57P 1138 1 44 284 1 44 107 1 44

CN 440 1 57 8456 1 57 082 1 57CP 374 1 44 892 1 44 002 1 44NP 324 1 44 1039 1 44 225 1 44

Root C 2339 1 90 2188 2 90 193 2 90N 469 1 90 3430 2 90 075 2 90P 338 1 65 1360 2 65 081 2 65

CN 009 1 90 6297 2 90 373 2 90CP 010 1 65 903 2 65 072 2 65NP 000 1 65 434 2 65 320 2 65

p lt 005 p lt 001 F indicates F value df denotes degree of freedom

For C gracilis the difference in the leaf N concentration and P concentration of stemand root between the two habitats was significant (p lt 005) the P concentrations of leafand the rootrsquos C and N concentrations in the epiphytic habitat significantly exceeded thosein terrestrial habitat (p lt 001) while the CN and CP in leaf between the two habitatsshowed a reverse trend (Table S1) For E monandrum the C and P concentration in leaf andthe C and N concentration and the CN ratio in root in the epiphytic habitat were higherthan those in terrestrial habitat (Table S2) For B longifolia the C N and P concentrations inleaf and root in the epiphytic habitat were higher than those in terrestrial habitat but viceversa for the CN and CP in leaf (Table S3) At the species level the plasticity of elementcomposition in epiphytic individuals of the three species were relatively higher than that ofterrestrial individuals expect C in root and P in stem of C gracilis C and N in leaf and rootof E monandrum and C and N in leaf and root CN in leaf of B longifolia (Tables S1ndashS3)

32 Variation of Stoichiometric Homeostasis among Different Species Organs and Elements inthe Field

For leaves the HN of C gracilis was weaker than either that of E monandrum orB longifolia while the latter was characterized by the strongest N homeostasis (Figure 2Table S4) The HP of species was ranked in the order of E monandrum gt C gracilis gt Blongifolia In terms of HNP it was strongest in C gracilis followed by E monandrum beingweakest in B longifolia For stems with the exception of NP E monandrum had H valuesfor N and P that were each stronger than those characterizing C gracilis For roots exceptfor HN the H values for P and NP descended among species in the order of B longifoliagt C gracilis gt E monandrum Whereas the corresponding order for HN was B longifolia gtE monandrum gt C gracilis For the mean values of H the difference of leaves and rootsamong three species and stems between two species were all not significant (Figure S1)

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Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stemand (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrumB longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species allstronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stemsand roots the mean values for HN and HP of the three species were all decreased in theorder of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems thehomeostasis for NP was stronger than the P element but weaker than the N element Inroots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gtHN gt HP)

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Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratiosin leaves stems and roots of three species Bars are the mean values with standard error Capital andlowercase letters indicate significant differences among organs and element types respectively

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment

In the cultivation experiment the concentration of N and P in leaves were significantlyaffected by species and treatment levels NP was significantly affected by the treatment level

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while the interaction between species and treatment level had no significant effects on theN and P concentration and their elemental ratio (Table 3) The N and P concentrations ofleaves were both increased as higher N and P concentrations were applied in solution For thestoichiometric homeostasis values of different elements of leaves in the two species all valueswere greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the Nelement was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their interactionon N and P concentrations and NP ratios in leaves of two species in the cultivation experiment

VariableSpecies Treatment Species times Level

F df p F df p F df p

N 22611 1 000 417 8 003 132 8 022P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098The numerator df are given in the table the denominator df is 8

Forests 2021 12 x FOR PEER REVIEW 9 of 15

treatment level while the interaction between species and treatment level had no signif-icant effects on the N and P concentration and their elemental ratio (Table 3) The N and P concentrations of leaves were both increased as higher N and P concentrations were ap-plied in solution For the stoichiometric homeostasis values of different elements of leaves in the two species all values were greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the N element was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their in-teraction on N and P concentrations and NP ratios in leaves of two species in the cultivation ex-periment

Variable Species Treatment Species times Level

F df p F df p F df p N 22611 1 000 417 8 003 132 8 022 P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098 The numerator df are given in the table the denominator df is 8

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N P and NP in the greenhouse cultivation experiment (a b c) H values for leaf N P and N P of Briggsia longifolia (d e f) H values for leaf N P and N P of Elatostema monandrum respectively

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N Pand NP in the greenhouse cultivation experiment (andashc) H values for leaf N P and N P of Briggsialongifolia (dndashf) H values for leaf N P and N P of Elatostema monandrum respectively

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4 Discussion41 Differences in Nutrient Use Strategy between Epiphytic and Terrestrial Individuals ofFacultative Epiphytes

In our field experiment the concentrations of N and P in epiphytic individuals ofthree species in the natural forest were higher than those in terrestrial individuals but CNCP and NP ratios in the former were lower than the latter (Tables S1ndashS3) This agreedwith the results from fern in our study area and bromeliads in humid montane forest andlowland forests elsewhere [134748] CN and CP ratios reflect the carbon fixation per unitnutrient the higher N and P concentration and lower CN and CP values are associatedwith luxury consumption of plants [164950] Accordingly in our study the use of N andP by epiphytic individuals were more luxurious than terrestrial individuals which agreeswith the result that terrestrial individuals of fern are more efficient in nutrient use thanepiphytic individuals [13] The plasticity in nutrient use strategy of facultative epiphytes isconsidered to be an adaptive mechanism to different environmental conditions in orderto exploit the available resources efficiently [1213] Our results indicated that facultativeepiphytes had more flexible plasticity in nutrient use strategies to adapt habitat shifts

Winkler and Zotz [51] found that epiphytes possessed lower nutrient use efficiencythan that of terrestrial plants Epiphytes are generally considered to survive in the nutrientintermittent epiphytic habitats through luxurious consumption of nutrients [12] Theyusually absorb large amounts of nutrients and store them in vacuoles when the nutrient isabundant [142949] Accordingly the lower N and P use of epiphytic individuals may berelated to the luxury uptake of N and P in epiphytic habitats Furthermore we found thatvariation in the elemental composition of epiphytic individuals exceeded that of terrestrialindividuals most likely because epiphytic habitats are more heterogeneous [552] and theelemental composition of plants is influenced by other environmental factors (eg wateravailability light intensity) besides soil alone [3849]

42 Effects of Nutrient Limitation on the Distribution of Three Facultative Epiphytes

The foliar NP ratio can be used as an indicator of the type of nutrient limitationon plant growth the lower NP ratios indicate the plants were limited by N [244953]whereas the higher NP ratios indicates P limitations were present [2954] Epiphytes areusually restricted by P [55] but in our research nutrient restriction types differ amongthe three species The NP ratio of terrestrial and epiphytic individuals of E monandrumand epiphytic individuals of B longifolia was lower than 12 (p-values = 0214 0526 0225respectively) (Tables S2 and S3) suggesting the growth of which was restricted by Navailability [29] Since the NP ratio in terrestrial and epiphytic individuals of C gracilisand terrestrial individuals of B longifolia was higher than 12 (p-values = 0004 0227 0009respectively) yet still lower than 16 (p-values = 0 005 0 0006 respectively) (Tables S1 andS3) we interpreted this to mean that the growth of which were limited by N or co-limited byN and P [28] Our results were consistent with the finding that facultative epiphytes on datepalms (Phoenix dactylifera) in southeastern Spain were subject to N-limited conditions [14]Furthermore in our research the lower NP ratio in epiphytic than terrestrial individualsimplied that N-restriction in the epiphytic habitat was more pronounced The higher Pconcentration in epiphytic individuals probably increases the demands for N thus makingthem more pronounced Furthermore the higher P concentration in plants can alleviate thedamage caused by drought stress [49] In this study the higher P concentration in epiphyticindividuals may reflect their adaptation to epiphytic habitats where water is intermittent

The factors that drive the distribution of facultative epiphytes are not clear butprevious results suggested that environmental factors can affect the distribution of epi-phytes [1446] For the three species in our research most individuals of E monandrum andB longifolia grow on the bark with mass mat or thick humus while the majority of C gracilisgrow on the forest floor For E monandrum and B longifolia the N-restriction in epiphytichabitats was stronger than terrestrial habitats therefore they may gradually shift to forestfloor to alleviate N limitation For C gracilis the type of nutrient limitation in terrestrial and

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epiphytic habitats was same this indicated that the nutrient status in epiphytic habitatscan satisfy its requirement Previous research indicated that the interspecific competition interrestrial habitats was more intense than that in epiphytic habitats [7] Therefore C gracilismay shift to epiphytic habitats to escape interspecific competition

43 Differences in Stoichiometric Homeostasis among Species Organs and Elements ofFacultative Epiphytes

Traditional theory predicts that the stoichiometric homeostasis of autotroph was rela-tively weak [1756] However mounting data suggest that plants have a strong regulatorycapacity for their element composition [202237] Our results of the field and greenhousecultivation experiment indicated that the values of HN HP and HNP of leaves were allgt4 (p = 0002 0004 respectively) (Figure 4 Table S4) both showed strong homeostasiswhich did not support our second hypothesis that the homeostasis of facultative epiphytesis weak These results for facultative epiphytes in our study are also at odds with previousresearch concluded that a weak homeostasis of plants implied higher ecological adapt-ability and better suitability to a more variable environment [33] This discrepancy maybe because that conclusion about relationship between stoichiometric homeostasis andecological adaptability of plants was almost based on studies of plant leaves [3349] Theelemental composition of leaves is insensitive to nutrient changes in the soil instead rootsare more valid indicators of nutrient change in the environment [203857ndash59] Therefore aleafrsquos stoichiometric homeostasis cannot accurately reflect the ecological adaptability ofplants [3860] In our work here although leaves did have strong stoichiometric home-ostasis it was rather weak for roots (Table S4) The main function of roots is to absorbmineral nutrients and water from the soil [33] their plastic nutrient absorption is helpfulfor facultative epiphytes to efficiently exploit the available resources in different habitatsThis result indicated that facultative epiphytes could adapt to variable environment bychanging the elemental composition of their roots

Moreover the difference in stoichiometric homeostasis among the three species in ourfield study was not significant and the stoichiometric homeostasis mainly affected by or-gans (Table S5) likely because all three share a similar species richness in the sampling areaand their ecological strategy and adaptability to the environment were comparable [22]This was also confirmed by the consistent response of the two species to nutrient additionin the greenhouse cultivation experiments in our study (Table 3)

Different organs of the same species exhibit disparate stoichiometric characteristicsto better adapt to the environment [162033] We found that stoichiometric homeostasiswas stronger in aboveground than belowground of the three facultative epiphytes Rootswere more sensitive to changes of elements concentrations in environmental resourceswhich agrees with findings reported for terrestrial grass and woody plants [21223138]However unlike the related studies on terrestrial woody plants [38] the stems of C gracilisand E monandrum show strong stoichiometric homeostasis (Table S4) This may be relatedto the insensitivity of some key elements in stems of facultative epiphyte to changes insoil nutrient concentrations The stems of woody and herbaceous plants take differentfunctions the stems of woody plants mainly play transportation and supporting rolewhile the stems of herbaceous plants have less structural C than woody plants yet bearthe functions of photosynthesis transportation and the supporting action [20] Thereforethe stems of herbaceous plants need relatively stable supply of elements to maintain theirphysiological functions

The strength of homeostasis is also related to the elemental concentration [61] Previ-ous research for terrestrial plants indicated that the homeostasis of macro-elements washigher than trace elements [3461] In our study the value of stoichiometric homeostasisof N and P in the three facultative epiphyte species showed the order of HN gt HP This isconsistent with results from vertebrates and trees with greater control over high-demandelements [34ndash36] Therefore the facultative epiphytic plants may also have greater con-trol over macro-elements highly demanded for physiology functioning However ourresearch only focused on the stoichiometric homeostasis of N and P elements in facultative

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epiphytes the stoichiometric homeostasis of trace elements remains unknown Thereforewhether the stoichiometric homeostasis of macro-elements in facultative epiphytes is higherthan trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumulationof N within a given plant tissue is usually accompanied by an increase in its P content thehomeostasis of NP is stronger than N and P elements [1762] However in our study thestoichiometric homeostasis value of NP in leaf and root were all higher than that of P butlower than that of N (Figure 3) Based on the theory that limiting elements should havestrong homeostasis [313463] we speculate that facultative epiphytes may be restricted byN availability These plants therefore would take strong regulation of N to cope with theunpredictable fluctuations of N elements in their habitat

5 Conclusions

This study systematically elucidates the nutrient use and ecological adaptation strate-gies of facultative epiphytes from the perspective of stoichiometry using field investigationand greenhouse cultivation experiments Our study showed that epiphytic individualsof facultative epiphytes survive intermittent habitat via luxury consumption of nutrientswhile terrestrial individuals use more conservative nutrients (Figure 5) The nutrient usestrategies of facultative epiphytes were flexible which enabled facultative epiphytes toexploit bark and soil interchangeably Facultative epiphytes had strong control over N andP concentrations and NP ratio of leaves and stems while roots had weaker stoichiometricstability These findings suggested that to adapt to the variable habitats in montane forestecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulatingthe absorption and composition of elements in roots

|

Forests 2021 12 x FOR PEER REVIEW 12 of 15

trol over macro-elements highly demanded for physiology functioning However our research only focused on the stoichiometric homeostasis of N and P elements in faculta-tive epiphytes the stoichiometric homeostasis of trace elements remains unknown Therefore whether the stoichiometric homeostasis of macro-elements in facultative epi-phytes is higher than trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumu-lation of N within a given plant tissue is usually accompanied by an increase in its P content the homeostasis of NP is stronger than N and P elements [1762] However in our study the stoichiometric homeostasis value of NP in leaf and root were all higher than that of P but lower than that of N (Figure 3) Based on the theory that limiting ele-ments should have strong homeostasis [313463] we speculate that facultative epiphytes may be restricted by N availability These plants therefore would take strong regulation of N to cope with the unpredictable fluctuations of N elements in their habitat

5 Conclusions This study systematically elucidates the nutrient use and ecological adaptation

strategies of facultative epiphytes from the perspective of stoichiometry using field in-vestigation and greenhouse cultivation experiments Our study showed that epiphytic individuals of facultative epiphytes survive intermittent habitat via luxury consumption of nutrients while terrestrial individuals use more conservative nutrients (Figure 5) The nutrient use strategies of facultative epiphytes were flexible which enabled facultative epiphytes to exploit bark and soil interchangeably Facultative epiphytes had strong control over N and P concentrations and NP ratio of leaves and stems while roots had weaker stoichiometric stability These findings suggested that to adapt to the variable habitats in montane forest ecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulating the absorption and composition of elements in roots

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic and terrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at wwwmdpicomxxxs1 Fig-ure S1 The mean stoichiometric homeostasis (H) values for N and P elements and their element ratios in leaf stem and root of different species Table S1 The difference of stoichiometric traits of C N and P concentrations and their element ratios in different organs of C gracilis between epi-

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic andterrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at httpswwwmdpicom1999-490712116s1 Figure S1 The mean stoichiometric homeostasis (H) values for N and Pelements and their element ratios in leaf stem and root of different species Table S1 The differenceof stoichiometric traits of C N and P concentrations and their element ratios in different organs of C

Forests 2021 12 16 7 of 15

Table 2 Effects of species habitats and their interactions on stoichiometric traits of C N and P indifferent organs of facultative epiphytes in field experiment

Organ VariableHabitat Species Habitat times Species

F df F df F df

Leaf C 367 1 93 63420 2 93 154 2 93N 456 1 93 9122 2 93 129 2 93P 1083 1 89 4399 2 89 301 2 89

CN 056 1 93 17012 2 93 110 2 93CP 773 1 89 9448 2 89 370 2 89NP 399 1 89 453 2 89 089 2 89

Stem C 706 1 57 20677 1 57 203 1 57N 143 1 57 9392 1 57 070 1 57P 1138 1 44 284 1 44 107 1 44

CN 440 1 57 8456 1 57 082 1 57CP 374 1 44 892 1 44 002 1 44NP 324 1 44 1039 1 44 225 1 44

Root C 2339 1 90 2188 2 90 193 2 90N 469 1 90 3430 2 90 075 2 90P 338 1 65 1360 2 65 081 2 65

CN 009 1 90 6297 2 90 373 2 90CP 010 1 65 903 2 65 072 2 65NP 000 1 65 434 2 65 320 2 65

p lt 005 p lt 001 F indicates F value df denotes degree of freedom

For C gracilis the difference in the leaf N concentration and P concentration of stemand root between the two habitats was significant (p lt 005) the P concentrations of leafand the rootrsquos C and N concentrations in the epiphytic habitat significantly exceeded thosein terrestrial habitat (p lt 001) while the CN and CP in leaf between the two habitatsshowed a reverse trend (Table S1) For E monandrum the C and P concentration in leaf andthe C and N concentration and the CN ratio in root in the epiphytic habitat were higherthan those in terrestrial habitat (Table S2) For B longifolia the C N and P concentrations inleaf and root in the epiphytic habitat were higher than those in terrestrial habitat but viceversa for the CN and CP in leaf (Table S3) At the species level the plasticity of elementcomposition in epiphytic individuals of the three species were relatively higher than that ofterrestrial individuals expect C in root and P in stem of C gracilis C and N in leaf and rootof E monandrum and C and N in leaf and root CN in leaf of B longifolia (Tables S1ndashS3)

32 Variation of Stoichiometric Homeostasis among Different Species Organs and Elements inthe Field

For leaves the HN of C gracilis was weaker than either that of E monandrum orB longifolia while the latter was characterized by the strongest N homeostasis (Figure 2Table S4) The HP of species was ranked in the order of E monandrum gt C gracilis gt Blongifolia In terms of HNP it was strongest in C gracilis followed by E monandrum beingweakest in B longifolia For stems with the exception of NP E monandrum had H valuesfor N and P that were each stronger than those characterizing C gracilis For roots exceptfor HN the H values for P and NP descended among species in the order of B longifoliagt C gracilis gt E monandrum Whereas the corresponding order for HN was B longifolia gtE monandrum gt C gracilis For the mean values of H the difference of leaves and rootsamong three species and stems between two species were all not significant (Figure S1)

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Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stemand (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrumB longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species allstronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stemsand roots the mean values for HN and HP of the three species were all decreased in theorder of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems thehomeostasis for NP was stronger than the P element but weaker than the N element Inroots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gtHN gt HP)

Forests 2021 12 x FOR PEER REVIEW 8 of 15

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratiosin leaves stems and roots of three species Bars are the mean values with standard error Capital andlowercase letters indicate significant differences among organs and element types respectively

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment

In the cultivation experiment the concentration of N and P in leaves were significantlyaffected by species and treatment levels NP was significantly affected by the treatment level

Forests 2021 12 16 9 of 15

while the interaction between species and treatment level had no significant effects on theN and P concentration and their elemental ratio (Table 3) The N and P concentrations ofleaves were both increased as higher N and P concentrations were applied in solution For thestoichiometric homeostasis values of different elements of leaves in the two species all valueswere greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the Nelement was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their interactionon N and P concentrations and NP ratios in leaves of two species in the cultivation experiment

VariableSpecies Treatment Species times Level

F df p F df p F df p

N 22611 1 000 417 8 003 132 8 022P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098The numerator df are given in the table the denominator df is 8

Forests 2021 12 x FOR PEER REVIEW 9 of 15

treatment level while the interaction between species and treatment level had no signif-icant effects on the N and P concentration and their elemental ratio (Table 3) The N and P concentrations of leaves were both increased as higher N and P concentrations were ap-plied in solution For the stoichiometric homeostasis values of different elements of leaves in the two species all values were greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the N element was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their in-teraction on N and P concentrations and NP ratios in leaves of two species in the cultivation ex-periment

Variable Species Treatment Species times Level

F df p F df p F df p N 22611 1 000 417 8 003 132 8 022 P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098 The numerator df are given in the table the denominator df is 8

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N P and NP in the greenhouse cultivation experiment (a b c) H values for leaf N P and N P of Briggsia longifolia (d e f) H values for leaf N P and N P of Elatostema monandrum respectively

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N Pand NP in the greenhouse cultivation experiment (andashc) H values for leaf N P and N P of Briggsialongifolia (dndashf) H values for leaf N P and N P of Elatostema monandrum respectively

Forests 2021 12 16 10 of 15

4 Discussion41 Differences in Nutrient Use Strategy between Epiphytic and Terrestrial Individuals ofFacultative Epiphytes

In our field experiment the concentrations of N and P in epiphytic individuals ofthree species in the natural forest were higher than those in terrestrial individuals but CNCP and NP ratios in the former were lower than the latter (Tables S1ndashS3) This agreedwith the results from fern in our study area and bromeliads in humid montane forest andlowland forests elsewhere [134748] CN and CP ratios reflect the carbon fixation per unitnutrient the higher N and P concentration and lower CN and CP values are associatedwith luxury consumption of plants [164950] Accordingly in our study the use of N andP by epiphytic individuals were more luxurious than terrestrial individuals which agreeswith the result that terrestrial individuals of fern are more efficient in nutrient use thanepiphytic individuals [13] The plasticity in nutrient use strategy of facultative epiphytes isconsidered to be an adaptive mechanism to different environmental conditions in orderto exploit the available resources efficiently [1213] Our results indicated that facultativeepiphytes had more flexible plasticity in nutrient use strategies to adapt habitat shifts

Winkler and Zotz [51] found that epiphytes possessed lower nutrient use efficiencythan that of terrestrial plants Epiphytes are generally considered to survive in the nutrientintermittent epiphytic habitats through luxurious consumption of nutrients [12] Theyusually absorb large amounts of nutrients and store them in vacuoles when the nutrient isabundant [142949] Accordingly the lower N and P use of epiphytic individuals may berelated to the luxury uptake of N and P in epiphytic habitats Furthermore we found thatvariation in the elemental composition of epiphytic individuals exceeded that of terrestrialindividuals most likely because epiphytic habitats are more heterogeneous [552] and theelemental composition of plants is influenced by other environmental factors (eg wateravailability light intensity) besides soil alone [3849]

42 Effects of Nutrient Limitation on the Distribution of Three Facultative Epiphytes

The foliar NP ratio can be used as an indicator of the type of nutrient limitationon plant growth the lower NP ratios indicate the plants were limited by N [244953]whereas the higher NP ratios indicates P limitations were present [2954] Epiphytes areusually restricted by P [55] but in our research nutrient restriction types differ amongthe three species The NP ratio of terrestrial and epiphytic individuals of E monandrumand epiphytic individuals of B longifolia was lower than 12 (p-values = 0214 0526 0225respectively) (Tables S2 and S3) suggesting the growth of which was restricted by Navailability [29] Since the NP ratio in terrestrial and epiphytic individuals of C gracilisand terrestrial individuals of B longifolia was higher than 12 (p-values = 0004 0227 0009respectively) yet still lower than 16 (p-values = 0 005 0 0006 respectively) (Tables S1 andS3) we interpreted this to mean that the growth of which were limited by N or co-limited byN and P [28] Our results were consistent with the finding that facultative epiphytes on datepalms (Phoenix dactylifera) in southeastern Spain were subject to N-limited conditions [14]Furthermore in our research the lower NP ratio in epiphytic than terrestrial individualsimplied that N-restriction in the epiphytic habitat was more pronounced The higher Pconcentration in epiphytic individuals probably increases the demands for N thus makingthem more pronounced Furthermore the higher P concentration in plants can alleviate thedamage caused by drought stress [49] In this study the higher P concentration in epiphyticindividuals may reflect their adaptation to epiphytic habitats where water is intermittent

The factors that drive the distribution of facultative epiphytes are not clear butprevious results suggested that environmental factors can affect the distribution of epi-phytes [1446] For the three species in our research most individuals of E monandrum andB longifolia grow on the bark with mass mat or thick humus while the majority of C gracilisgrow on the forest floor For E monandrum and B longifolia the N-restriction in epiphytichabitats was stronger than terrestrial habitats therefore they may gradually shift to forestfloor to alleviate N limitation For C gracilis the type of nutrient limitation in terrestrial and

Forests 2021 12 16 11 of 15

epiphytic habitats was same this indicated that the nutrient status in epiphytic habitatscan satisfy its requirement Previous research indicated that the interspecific competition interrestrial habitats was more intense than that in epiphytic habitats [7] Therefore C gracilismay shift to epiphytic habitats to escape interspecific competition

43 Differences in Stoichiometric Homeostasis among Species Organs and Elements ofFacultative Epiphytes

Traditional theory predicts that the stoichiometric homeostasis of autotroph was rela-tively weak [1756] However mounting data suggest that plants have a strong regulatorycapacity for their element composition [202237] Our results of the field and greenhousecultivation experiment indicated that the values of HN HP and HNP of leaves were allgt4 (p = 0002 0004 respectively) (Figure 4 Table S4) both showed strong homeostasiswhich did not support our second hypothesis that the homeostasis of facultative epiphytesis weak These results for facultative epiphytes in our study are also at odds with previousresearch concluded that a weak homeostasis of plants implied higher ecological adapt-ability and better suitability to a more variable environment [33] This discrepancy maybe because that conclusion about relationship between stoichiometric homeostasis andecological adaptability of plants was almost based on studies of plant leaves [3349] Theelemental composition of leaves is insensitive to nutrient changes in the soil instead rootsare more valid indicators of nutrient change in the environment [203857ndash59] Therefore aleafrsquos stoichiometric homeostasis cannot accurately reflect the ecological adaptability ofplants [3860] In our work here although leaves did have strong stoichiometric home-ostasis it was rather weak for roots (Table S4) The main function of roots is to absorbmineral nutrients and water from the soil [33] their plastic nutrient absorption is helpfulfor facultative epiphytes to efficiently exploit the available resources in different habitatsThis result indicated that facultative epiphytes could adapt to variable environment bychanging the elemental composition of their roots

Moreover the difference in stoichiometric homeostasis among the three species in ourfield study was not significant and the stoichiometric homeostasis mainly affected by or-gans (Table S5) likely because all three share a similar species richness in the sampling areaand their ecological strategy and adaptability to the environment were comparable [22]This was also confirmed by the consistent response of the two species to nutrient additionin the greenhouse cultivation experiments in our study (Table 3)

Different organs of the same species exhibit disparate stoichiometric characteristicsto better adapt to the environment [162033] We found that stoichiometric homeostasiswas stronger in aboveground than belowground of the three facultative epiphytes Rootswere more sensitive to changes of elements concentrations in environmental resourceswhich agrees with findings reported for terrestrial grass and woody plants [21223138]However unlike the related studies on terrestrial woody plants [38] the stems of C gracilisand E monandrum show strong stoichiometric homeostasis (Table S4) This may be relatedto the insensitivity of some key elements in stems of facultative epiphyte to changes insoil nutrient concentrations The stems of woody and herbaceous plants take differentfunctions the stems of woody plants mainly play transportation and supporting rolewhile the stems of herbaceous plants have less structural C than woody plants yet bearthe functions of photosynthesis transportation and the supporting action [20] Thereforethe stems of herbaceous plants need relatively stable supply of elements to maintain theirphysiological functions

The strength of homeostasis is also related to the elemental concentration [61] Previ-ous research for terrestrial plants indicated that the homeostasis of macro-elements washigher than trace elements [3461] In our study the value of stoichiometric homeostasisof N and P in the three facultative epiphyte species showed the order of HN gt HP This isconsistent with results from vertebrates and trees with greater control over high-demandelements [34ndash36] Therefore the facultative epiphytic plants may also have greater con-trol over macro-elements highly demanded for physiology functioning However ourresearch only focused on the stoichiometric homeostasis of N and P elements in facultative

Forests 2021 12 16 12 of 15

epiphytes the stoichiometric homeostasis of trace elements remains unknown Thereforewhether the stoichiometric homeostasis of macro-elements in facultative epiphytes is higherthan trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumulationof N within a given plant tissue is usually accompanied by an increase in its P content thehomeostasis of NP is stronger than N and P elements [1762] However in our study thestoichiometric homeostasis value of NP in leaf and root were all higher than that of P butlower than that of N (Figure 3) Based on the theory that limiting elements should havestrong homeostasis [313463] we speculate that facultative epiphytes may be restricted byN availability These plants therefore would take strong regulation of N to cope with theunpredictable fluctuations of N elements in their habitat

5 Conclusions

This study systematically elucidates the nutrient use and ecological adaptation strate-gies of facultative epiphytes from the perspective of stoichiometry using field investigationand greenhouse cultivation experiments Our study showed that epiphytic individualsof facultative epiphytes survive intermittent habitat via luxury consumption of nutrientswhile terrestrial individuals use more conservative nutrients (Figure 5) The nutrient usestrategies of facultative epiphytes were flexible which enabled facultative epiphytes toexploit bark and soil interchangeably Facultative epiphytes had strong control over N andP concentrations and NP ratio of leaves and stems while roots had weaker stoichiometricstability These findings suggested that to adapt to the variable habitats in montane forestecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulatingthe absorption and composition of elements in roots

|

Forests 2021 12 x FOR PEER REVIEW 12 of 15

trol over macro-elements highly demanded for physiology functioning However our research only focused on the stoichiometric homeostasis of N and P elements in faculta-tive epiphytes the stoichiometric homeostasis of trace elements remains unknown Therefore whether the stoichiometric homeostasis of macro-elements in facultative epi-phytes is higher than trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumu-lation of N within a given plant tissue is usually accompanied by an increase in its P content the homeostasis of NP is stronger than N and P elements [1762] However in our study the stoichiometric homeostasis value of NP in leaf and root were all higher than that of P but lower than that of N (Figure 3) Based on the theory that limiting ele-ments should have strong homeostasis [313463] we speculate that facultative epiphytes may be restricted by N availability These plants therefore would take strong regulation of N to cope with the unpredictable fluctuations of N elements in their habitat

5 Conclusions This study systematically elucidates the nutrient use and ecological adaptation

strategies of facultative epiphytes from the perspective of stoichiometry using field in-vestigation and greenhouse cultivation experiments Our study showed that epiphytic individuals of facultative epiphytes survive intermittent habitat via luxury consumption of nutrients while terrestrial individuals use more conservative nutrients (Figure 5) The nutrient use strategies of facultative epiphytes were flexible which enabled facultative epiphytes to exploit bark and soil interchangeably Facultative epiphytes had strong control over N and P concentrations and NP ratio of leaves and stems while roots had weaker stoichiometric stability These findings suggested that to adapt to the variable habitats in montane forest ecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulating the absorption and composition of elements in roots

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic and terrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at wwwmdpicomxxxs1 Fig-ure S1 The mean stoichiometric homeostasis (H) values for N and P elements and their element ratios in leaf stem and root of different species Table S1 The difference of stoichiometric traits of C N and P concentrations and their element ratios in different organs of C gracilis between epi-

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic andterrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at httpswwwmdpicom1999-490712116s1 Figure S1 The mean stoichiometric homeostasis (H) values for N and Pelements and their element ratios in leaf stem and root of different species Table S1 The differenceof stoichiometric traits of C N and P concentrations and their element ratios in different organs of C

Forests 2021 12 16 8 of 15

Forests 2021 12 x FOR PEER REVIEW 8 of 15

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stemand (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrumB longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species allstronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stemsand roots the mean values for HN and HP of the three species were all decreased in theorder of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems thehomeostasis for NP was stronger than the P element but weaker than the N element Inroots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gtHN gt HP)

Forests 2021 12 x FOR PEER REVIEW 8 of 15

Figure 2 The stoichiometric homeostasis (H) values for N and P elements and their element ratios in the (A) leaf (B) stem and (C) root parts of different species CAGR Cautleya gracilis BRLO Briggsia longifolia ELMO Elatostema monandrum B longifolia has no stem and thus only stems of C gracilis and E monandrum were analyzed

The mean value of H for N and P elements in leaves and stems of three species all stronger than their roots while HNP decreased in the order of leaf gt root gt stem (Figure 3)

Although the differences between HN and HP were insignificant for leaves stems and roots the mean values for HN and HP of the three species were all decreased in the order of HN gt HP (Figure 3) In leaves HNP exceeded either HN or HP In stems the home-ostasis for NP was stronger than the P element but weaker than the N element In roots the rank order of HN HP and HNP was consistent with that for leaves (ie HNP gt HN gt HP)

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratios in leaves stems and roots of three species Bars are the mean values with standard error Capital and lowercase letters indicate significant differences among organs and element types respective-ly

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment In the cultivation experiment the concentration of N and P in leaves were signifi-

cantly affected by species and treatment levels NP was significantly affected by the

Figure 3 The mean stoichiometric homeostasis (H) values for the N and P elements and their ratiosin leaves stems and roots of three species Bars are the mean values with standard error Capital andlowercase letters indicate significant differences among organs and element types respectively

33 Stoichiometric Homeostasis of Leaf N P and NP in Greenhouse Cultivation Experiment

In the cultivation experiment the concentration of N and P in leaves were significantlyaffected by species and treatment levels NP was significantly affected by the treatment level

Forests 2021 12 16 9 of 15

while the interaction between species and treatment level had no significant effects on theN and P concentration and their elemental ratio (Table 3) The N and P concentrations ofleaves were both increased as higher N and P concentrations were applied in solution For thestoichiometric homeostasis values of different elements of leaves in the two species all valueswere greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the Nelement was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their interactionon N and P concentrations and NP ratios in leaves of two species in the cultivation experiment

VariableSpecies Treatment Species times Level

F df p F df p F df p

N 22611 1 000 417 8 003 132 8 022P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098The numerator df are given in the table the denominator df is 8

Forests 2021 12 x FOR PEER REVIEW 9 of 15

treatment level while the interaction between species and treatment level had no signif-icant effects on the N and P concentration and their elemental ratio (Table 3) The N and P concentrations of leaves were both increased as higher N and P concentrations were ap-plied in solution For the stoichiometric homeostasis values of different elements of leaves in the two species all values were greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the N element was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their in-teraction on N and P concentrations and NP ratios in leaves of two species in the cultivation ex-periment

Variable Species Treatment Species times Level

F df p F df p F df p N 22611 1 000 417 8 003 132 8 022 P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098 The numerator df are given in the table the denominator df is 8

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N P and NP in the greenhouse cultivation experiment (a b c) H values for leaf N P and N P of Briggsia longifolia (d e f) H values for leaf N P and N P of Elatostema monandrum respectively

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N Pand NP in the greenhouse cultivation experiment (andashc) H values for leaf N P and N P of Briggsialongifolia (dndashf) H values for leaf N P and N P of Elatostema monandrum respectively

Forests 2021 12 16 10 of 15

4 Discussion41 Differences in Nutrient Use Strategy between Epiphytic and Terrestrial Individuals ofFacultative Epiphytes

In our field experiment the concentrations of N and P in epiphytic individuals ofthree species in the natural forest were higher than those in terrestrial individuals but CNCP and NP ratios in the former were lower than the latter (Tables S1ndashS3) This agreedwith the results from fern in our study area and bromeliads in humid montane forest andlowland forests elsewhere [134748] CN and CP ratios reflect the carbon fixation per unitnutrient the higher N and P concentration and lower CN and CP values are associatedwith luxury consumption of plants [164950] Accordingly in our study the use of N andP by epiphytic individuals were more luxurious than terrestrial individuals which agreeswith the result that terrestrial individuals of fern are more efficient in nutrient use thanepiphytic individuals [13] The plasticity in nutrient use strategy of facultative epiphytes isconsidered to be an adaptive mechanism to different environmental conditions in orderto exploit the available resources efficiently [1213] Our results indicated that facultativeepiphytes had more flexible plasticity in nutrient use strategies to adapt habitat shifts

Winkler and Zotz [51] found that epiphytes possessed lower nutrient use efficiencythan that of terrestrial plants Epiphytes are generally considered to survive in the nutrientintermittent epiphytic habitats through luxurious consumption of nutrients [12] Theyusually absorb large amounts of nutrients and store them in vacuoles when the nutrient isabundant [142949] Accordingly the lower N and P use of epiphytic individuals may berelated to the luxury uptake of N and P in epiphytic habitats Furthermore we found thatvariation in the elemental composition of epiphytic individuals exceeded that of terrestrialindividuals most likely because epiphytic habitats are more heterogeneous [552] and theelemental composition of plants is influenced by other environmental factors (eg wateravailability light intensity) besides soil alone [3849]

42 Effects of Nutrient Limitation on the Distribution of Three Facultative Epiphytes

The foliar NP ratio can be used as an indicator of the type of nutrient limitationon plant growth the lower NP ratios indicate the plants were limited by N [244953]whereas the higher NP ratios indicates P limitations were present [2954] Epiphytes areusually restricted by P [55] but in our research nutrient restriction types differ amongthe three species The NP ratio of terrestrial and epiphytic individuals of E monandrumand epiphytic individuals of B longifolia was lower than 12 (p-values = 0214 0526 0225respectively) (Tables S2 and S3) suggesting the growth of which was restricted by Navailability [29] Since the NP ratio in terrestrial and epiphytic individuals of C gracilisand terrestrial individuals of B longifolia was higher than 12 (p-values = 0004 0227 0009respectively) yet still lower than 16 (p-values = 0 005 0 0006 respectively) (Tables S1 andS3) we interpreted this to mean that the growth of which were limited by N or co-limited byN and P [28] Our results were consistent with the finding that facultative epiphytes on datepalms (Phoenix dactylifera) in southeastern Spain were subject to N-limited conditions [14]Furthermore in our research the lower NP ratio in epiphytic than terrestrial individualsimplied that N-restriction in the epiphytic habitat was more pronounced The higher Pconcentration in epiphytic individuals probably increases the demands for N thus makingthem more pronounced Furthermore the higher P concentration in plants can alleviate thedamage caused by drought stress [49] In this study the higher P concentration in epiphyticindividuals may reflect their adaptation to epiphytic habitats where water is intermittent

The factors that drive the distribution of facultative epiphytes are not clear butprevious results suggested that environmental factors can affect the distribution of epi-phytes [1446] For the three species in our research most individuals of E monandrum andB longifolia grow on the bark with mass mat or thick humus while the majority of C gracilisgrow on the forest floor For E monandrum and B longifolia the N-restriction in epiphytichabitats was stronger than terrestrial habitats therefore they may gradually shift to forestfloor to alleviate N limitation For C gracilis the type of nutrient limitation in terrestrial and

Forests 2021 12 16 11 of 15

epiphytic habitats was same this indicated that the nutrient status in epiphytic habitatscan satisfy its requirement Previous research indicated that the interspecific competition interrestrial habitats was more intense than that in epiphytic habitats [7] Therefore C gracilismay shift to epiphytic habitats to escape interspecific competition

43 Differences in Stoichiometric Homeostasis among Species Organs and Elements ofFacultative Epiphytes

Traditional theory predicts that the stoichiometric homeostasis of autotroph was rela-tively weak [1756] However mounting data suggest that plants have a strong regulatorycapacity for their element composition [202237] Our results of the field and greenhousecultivation experiment indicated that the values of HN HP and HNP of leaves were allgt4 (p = 0002 0004 respectively) (Figure 4 Table S4) both showed strong homeostasiswhich did not support our second hypothesis that the homeostasis of facultative epiphytesis weak These results for facultative epiphytes in our study are also at odds with previousresearch concluded that a weak homeostasis of plants implied higher ecological adapt-ability and better suitability to a more variable environment [33] This discrepancy maybe because that conclusion about relationship between stoichiometric homeostasis andecological adaptability of plants was almost based on studies of plant leaves [3349] Theelemental composition of leaves is insensitive to nutrient changes in the soil instead rootsare more valid indicators of nutrient change in the environment [203857ndash59] Therefore aleafrsquos stoichiometric homeostasis cannot accurately reflect the ecological adaptability ofplants [3860] In our work here although leaves did have strong stoichiometric home-ostasis it was rather weak for roots (Table S4) The main function of roots is to absorbmineral nutrients and water from the soil [33] their plastic nutrient absorption is helpfulfor facultative epiphytes to efficiently exploit the available resources in different habitatsThis result indicated that facultative epiphytes could adapt to variable environment bychanging the elemental composition of their roots

Moreover the difference in stoichiometric homeostasis among the three species in ourfield study was not significant and the stoichiometric homeostasis mainly affected by or-gans (Table S5) likely because all three share a similar species richness in the sampling areaand their ecological strategy and adaptability to the environment were comparable [22]This was also confirmed by the consistent response of the two species to nutrient additionin the greenhouse cultivation experiments in our study (Table 3)

Different organs of the same species exhibit disparate stoichiometric characteristicsto better adapt to the environment [162033] We found that stoichiometric homeostasiswas stronger in aboveground than belowground of the three facultative epiphytes Rootswere more sensitive to changes of elements concentrations in environmental resourceswhich agrees with findings reported for terrestrial grass and woody plants [21223138]However unlike the related studies on terrestrial woody plants [38] the stems of C gracilisand E monandrum show strong stoichiometric homeostasis (Table S4) This may be relatedto the insensitivity of some key elements in stems of facultative epiphyte to changes insoil nutrient concentrations The stems of woody and herbaceous plants take differentfunctions the stems of woody plants mainly play transportation and supporting rolewhile the stems of herbaceous plants have less structural C than woody plants yet bearthe functions of photosynthesis transportation and the supporting action [20] Thereforethe stems of herbaceous plants need relatively stable supply of elements to maintain theirphysiological functions

The strength of homeostasis is also related to the elemental concentration [61] Previ-ous research for terrestrial plants indicated that the homeostasis of macro-elements washigher than trace elements [3461] In our study the value of stoichiometric homeostasisof N and P in the three facultative epiphyte species showed the order of HN gt HP This isconsistent with results from vertebrates and trees with greater control over high-demandelements [34ndash36] Therefore the facultative epiphytic plants may also have greater con-trol over macro-elements highly demanded for physiology functioning However ourresearch only focused on the stoichiometric homeostasis of N and P elements in facultative

Forests 2021 12 16 12 of 15

epiphytes the stoichiometric homeostasis of trace elements remains unknown Thereforewhether the stoichiometric homeostasis of macro-elements in facultative epiphytes is higherthan trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumulationof N within a given plant tissue is usually accompanied by an increase in its P content thehomeostasis of NP is stronger than N and P elements [1762] However in our study thestoichiometric homeostasis value of NP in leaf and root were all higher than that of P butlower than that of N (Figure 3) Based on the theory that limiting elements should havestrong homeostasis [313463] we speculate that facultative epiphytes may be restricted byN availability These plants therefore would take strong regulation of N to cope with theunpredictable fluctuations of N elements in their habitat

5 Conclusions

This study systematically elucidates the nutrient use and ecological adaptation strate-gies of facultative epiphytes from the perspective of stoichiometry using field investigationand greenhouse cultivation experiments Our study showed that epiphytic individualsof facultative epiphytes survive intermittent habitat via luxury consumption of nutrientswhile terrestrial individuals use more conservative nutrients (Figure 5) The nutrient usestrategies of facultative epiphytes were flexible which enabled facultative epiphytes toexploit bark and soil interchangeably Facultative epiphytes had strong control over N andP concentrations and NP ratio of leaves and stems while roots had weaker stoichiometricstability These findings suggested that to adapt to the variable habitats in montane forestecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulatingthe absorption and composition of elements in roots

|

Forests 2021 12 x FOR PEER REVIEW 12 of 15

trol over macro-elements highly demanded for physiology functioning However our research only focused on the stoichiometric homeostasis of N and P elements in faculta-tive epiphytes the stoichiometric homeostasis of trace elements remains unknown Therefore whether the stoichiometric homeostasis of macro-elements in facultative epi-phytes is higher than trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumu-lation of N within a given plant tissue is usually accompanied by an increase in its P content the homeostasis of NP is stronger than N and P elements [1762] However in our study the stoichiometric homeostasis value of NP in leaf and root were all higher than that of P but lower than that of N (Figure 3) Based on the theory that limiting ele-ments should have strong homeostasis [313463] we speculate that facultative epiphytes may be restricted by N availability These plants therefore would take strong regulation of N to cope with the unpredictable fluctuations of N elements in their habitat

5 Conclusions This study systematically elucidates the nutrient use and ecological adaptation

strategies of facultative epiphytes from the perspective of stoichiometry using field in-vestigation and greenhouse cultivation experiments Our study showed that epiphytic individuals of facultative epiphytes survive intermittent habitat via luxury consumption of nutrients while terrestrial individuals use more conservative nutrients (Figure 5) The nutrient use strategies of facultative epiphytes were flexible which enabled facultative epiphytes to exploit bark and soil interchangeably Facultative epiphytes had strong control over N and P concentrations and NP ratio of leaves and stems while roots had weaker stoichiometric stability These findings suggested that to adapt to the variable habitats in montane forest ecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulating the absorption and composition of elements in roots

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic and terrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at wwwmdpicomxxxs1 Fig-ure S1 The mean stoichiometric homeostasis (H) values for N and P elements and their element ratios in leaf stem and root of different species Table S1 The difference of stoichiometric traits of C N and P concentrations and their element ratios in different organs of C gracilis between epi-

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic andterrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at httpswwwmdpicom1999-490712116s1 Figure S1 The mean stoichiometric homeostasis (H) values for N and Pelements and their element ratios in leaf stem and root of different species Table S1 The differenceof stoichiometric traits of C N and P concentrations and their element ratios in different organs of C

Forests 2021 12 16 9 of 15

while the interaction between species and treatment level had no significant effects on theN and P concentration and their elemental ratio (Table 3) The N and P concentrations ofleaves were both increased as higher N and P concentrations were applied in solution For thestoichiometric homeostasis values of different elements of leaves in the two species all valueswere greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the Nelement was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their interactionon N and P concentrations and NP ratios in leaves of two species in the cultivation experiment

VariableSpecies Treatment Species times Level

F df p F df p F df p

N 22611 1 000 417 8 003 132 8 022P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098The numerator df are given in the table the denominator df is 8

Forests 2021 12 x FOR PEER REVIEW 9 of 15

treatment level while the interaction between species and treatment level had no signif-icant effects on the N and P concentration and their elemental ratio (Table 3) The N and P concentrations of leaves were both increased as higher N and P concentrations were ap-plied in solution For the stoichiometric homeostasis values of different elements of leaves in the two species all values were greater than 4 (p = 0002) which indicated a state of strict homeostasis for which the N element was under stronger homeostasis than the P element (p = 0461) (Figure 4)

Table 3 Results of two-way ANOVA of the effects of the species and treatment level and their in-teraction on N and P concentrations and NP ratios in leaves of two species in the cultivation ex-periment

Variable Species Treatment Species times Level

F df p F df p F df p N 22611 1 000 417 8 003 132 8 022 P 24134 1 000 494 8 002 197 8 018

NP 895 1 002 073 8 067 020 8 098 The numerator df are given in the table the denominator df is 8

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N P and NP in the greenhouse cultivation experiment (a b c) H values for leaf N P and N P of Briggsia longifolia (d e f) H values for leaf N P and N P of Elatostema monandrum respectively

Figure 4 The statistics of homeostasis model and stoichiometric homeostasis (H) values for leaf N Pand NP in the greenhouse cultivation experiment (andashc) H values for leaf N P and N P of Briggsialongifolia (dndashf) H values for leaf N P and N P of Elatostema monandrum respectively

Forests 2021 12 16 10 of 15

4 Discussion41 Differences in Nutrient Use Strategy between Epiphytic and Terrestrial Individuals ofFacultative Epiphytes

In our field experiment the concentrations of N and P in epiphytic individuals ofthree species in the natural forest were higher than those in terrestrial individuals but CNCP and NP ratios in the former were lower than the latter (Tables S1ndashS3) This agreedwith the results from fern in our study area and bromeliads in humid montane forest andlowland forests elsewhere [134748] CN and CP ratios reflect the carbon fixation per unitnutrient the higher N and P concentration and lower CN and CP values are associatedwith luxury consumption of plants [164950] Accordingly in our study the use of N andP by epiphytic individuals were more luxurious than terrestrial individuals which agreeswith the result that terrestrial individuals of fern are more efficient in nutrient use thanepiphytic individuals [13] The plasticity in nutrient use strategy of facultative epiphytes isconsidered to be an adaptive mechanism to different environmental conditions in orderto exploit the available resources efficiently [1213] Our results indicated that facultativeepiphytes had more flexible plasticity in nutrient use strategies to adapt habitat shifts

Winkler and Zotz [51] found that epiphytes possessed lower nutrient use efficiencythan that of terrestrial plants Epiphytes are generally considered to survive in the nutrientintermittent epiphytic habitats through luxurious consumption of nutrients [12] Theyusually absorb large amounts of nutrients and store them in vacuoles when the nutrient isabundant [142949] Accordingly the lower N and P use of epiphytic individuals may berelated to the luxury uptake of N and P in epiphytic habitats Furthermore we found thatvariation in the elemental composition of epiphytic individuals exceeded that of terrestrialindividuals most likely because epiphytic habitats are more heterogeneous [552] and theelemental composition of plants is influenced by other environmental factors (eg wateravailability light intensity) besides soil alone [3849]

42 Effects of Nutrient Limitation on the Distribution of Three Facultative Epiphytes

The foliar NP ratio can be used as an indicator of the type of nutrient limitationon plant growth the lower NP ratios indicate the plants were limited by N [244953]whereas the higher NP ratios indicates P limitations were present [2954] Epiphytes areusually restricted by P [55] but in our research nutrient restriction types differ amongthe three species The NP ratio of terrestrial and epiphytic individuals of E monandrumand epiphytic individuals of B longifolia was lower than 12 (p-values = 0214 0526 0225respectively) (Tables S2 and S3) suggesting the growth of which was restricted by Navailability [29] Since the NP ratio in terrestrial and epiphytic individuals of C gracilisand terrestrial individuals of B longifolia was higher than 12 (p-values = 0004 0227 0009respectively) yet still lower than 16 (p-values = 0 005 0 0006 respectively) (Tables S1 andS3) we interpreted this to mean that the growth of which were limited by N or co-limited byN and P [28] Our results were consistent with the finding that facultative epiphytes on datepalms (Phoenix dactylifera) in southeastern Spain were subject to N-limited conditions [14]Furthermore in our research the lower NP ratio in epiphytic than terrestrial individualsimplied that N-restriction in the epiphytic habitat was more pronounced The higher Pconcentration in epiphytic individuals probably increases the demands for N thus makingthem more pronounced Furthermore the higher P concentration in plants can alleviate thedamage caused by drought stress [49] In this study the higher P concentration in epiphyticindividuals may reflect their adaptation to epiphytic habitats where water is intermittent

The factors that drive the distribution of facultative epiphytes are not clear butprevious results suggested that environmental factors can affect the distribution of epi-phytes [1446] For the three species in our research most individuals of E monandrum andB longifolia grow on the bark with mass mat or thick humus while the majority of C gracilisgrow on the forest floor For E monandrum and B longifolia the N-restriction in epiphytichabitats was stronger than terrestrial habitats therefore they may gradually shift to forestfloor to alleviate N limitation For C gracilis the type of nutrient limitation in terrestrial and

Forests 2021 12 16 11 of 15

epiphytic habitats was same this indicated that the nutrient status in epiphytic habitatscan satisfy its requirement Previous research indicated that the interspecific competition interrestrial habitats was more intense than that in epiphytic habitats [7] Therefore C gracilismay shift to epiphytic habitats to escape interspecific competition

43 Differences in Stoichiometric Homeostasis among Species Organs and Elements ofFacultative Epiphytes

Traditional theory predicts that the stoichiometric homeostasis of autotroph was rela-tively weak [1756] However mounting data suggest that plants have a strong regulatorycapacity for their element composition [202237] Our results of the field and greenhousecultivation experiment indicated that the values of HN HP and HNP of leaves were allgt4 (p = 0002 0004 respectively) (Figure 4 Table S4) both showed strong homeostasiswhich did not support our second hypothesis that the homeostasis of facultative epiphytesis weak These results for facultative epiphytes in our study are also at odds with previousresearch concluded that a weak homeostasis of plants implied higher ecological adapt-ability and better suitability to a more variable environment [33] This discrepancy maybe because that conclusion about relationship between stoichiometric homeostasis andecological adaptability of plants was almost based on studies of plant leaves [3349] Theelemental composition of leaves is insensitive to nutrient changes in the soil instead rootsare more valid indicators of nutrient change in the environment [203857ndash59] Therefore aleafrsquos stoichiometric homeostasis cannot accurately reflect the ecological adaptability ofplants [3860] In our work here although leaves did have strong stoichiometric home-ostasis it was rather weak for roots (Table S4) The main function of roots is to absorbmineral nutrients and water from the soil [33] their plastic nutrient absorption is helpfulfor facultative epiphytes to efficiently exploit the available resources in different habitatsThis result indicated that facultative epiphytes could adapt to variable environment bychanging the elemental composition of their roots

Moreover the difference in stoichiometric homeostasis among the three species in ourfield study was not significant and the stoichiometric homeostasis mainly affected by or-gans (Table S5) likely because all three share a similar species richness in the sampling areaand their ecological strategy and adaptability to the environment were comparable [22]This was also confirmed by the consistent response of the two species to nutrient additionin the greenhouse cultivation experiments in our study (Table 3)

Different organs of the same species exhibit disparate stoichiometric characteristicsto better adapt to the environment [162033] We found that stoichiometric homeostasiswas stronger in aboveground than belowground of the three facultative epiphytes Rootswere more sensitive to changes of elements concentrations in environmental resourceswhich agrees with findings reported for terrestrial grass and woody plants [21223138]However unlike the related studies on terrestrial woody plants [38] the stems of C gracilisand E monandrum show strong stoichiometric homeostasis (Table S4) This may be relatedto the insensitivity of some key elements in stems of facultative epiphyte to changes insoil nutrient concentrations The stems of woody and herbaceous plants take differentfunctions the stems of woody plants mainly play transportation and supporting rolewhile the stems of herbaceous plants have less structural C than woody plants yet bearthe functions of photosynthesis transportation and the supporting action [20] Thereforethe stems of herbaceous plants need relatively stable supply of elements to maintain theirphysiological functions

The strength of homeostasis is also related to the elemental concentration [61] Previ-ous research for terrestrial plants indicated that the homeostasis of macro-elements washigher than trace elements [3461] In our study the value of stoichiometric homeostasisof N and P in the three facultative epiphyte species showed the order of HN gt HP This isconsistent with results from vertebrates and trees with greater control over high-demandelements [34ndash36] Therefore the facultative epiphytic plants may also have greater con-trol over macro-elements highly demanded for physiology functioning However ourresearch only focused on the stoichiometric homeostasis of N and P elements in facultative

Forests 2021 12 16 12 of 15

epiphytes the stoichiometric homeostasis of trace elements remains unknown Thereforewhether the stoichiometric homeostasis of macro-elements in facultative epiphytes is higherthan trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumulationof N within a given plant tissue is usually accompanied by an increase in its P content thehomeostasis of NP is stronger than N and P elements [1762] However in our study thestoichiometric homeostasis value of NP in leaf and root were all higher than that of P butlower than that of N (Figure 3) Based on the theory that limiting elements should havestrong homeostasis [313463] we speculate that facultative epiphytes may be restricted byN availability These plants therefore would take strong regulation of N to cope with theunpredictable fluctuations of N elements in their habitat

5 Conclusions

This study systematically elucidates the nutrient use and ecological adaptation strate-gies of facultative epiphytes from the perspective of stoichiometry using field investigationand greenhouse cultivation experiments Our study showed that epiphytic individualsof facultative epiphytes survive intermittent habitat via luxury consumption of nutrientswhile terrestrial individuals use more conservative nutrients (Figure 5) The nutrient usestrategies of facultative epiphytes were flexible which enabled facultative epiphytes toexploit bark and soil interchangeably Facultative epiphytes had strong control over N andP concentrations and NP ratio of leaves and stems while roots had weaker stoichiometricstability These findings suggested that to adapt to the variable habitats in montane forestecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulatingthe absorption and composition of elements in roots

|

Forests 2021 12 x FOR PEER REVIEW 12 of 15

trol over macro-elements highly demanded for physiology functioning However our research only focused on the stoichiometric homeostasis of N and P elements in faculta-tive epiphytes the stoichiometric homeostasis of trace elements remains unknown Therefore whether the stoichiometric homeostasis of macro-elements in facultative epi-phytes is higher than trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumu-lation of N within a given plant tissue is usually accompanied by an increase in its P content the homeostasis of NP is stronger than N and P elements [1762] However in our study the stoichiometric homeostasis value of NP in leaf and root were all higher than that of P but lower than that of N (Figure 3) Based on the theory that limiting ele-ments should have strong homeostasis [313463] we speculate that facultative epiphytes may be restricted by N availability These plants therefore would take strong regulation of N to cope with the unpredictable fluctuations of N elements in their habitat

5 Conclusions This study systematically elucidates the nutrient use and ecological adaptation

strategies of facultative epiphytes from the perspective of stoichiometry using field in-vestigation and greenhouse cultivation experiments Our study showed that epiphytic individuals of facultative epiphytes survive intermittent habitat via luxury consumption of nutrients while terrestrial individuals use more conservative nutrients (Figure 5) The nutrient use strategies of facultative epiphytes were flexible which enabled facultative epiphytes to exploit bark and soil interchangeably Facultative epiphytes had strong control over N and P concentrations and NP ratio of leaves and stems while roots had weaker stoichiometric stability These findings suggested that to adapt to the variable habitats in montane forest ecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulating the absorption and composition of elements in roots

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic and terrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at wwwmdpicomxxxs1 Fig-ure S1 The mean stoichiometric homeostasis (H) values for N and P elements and their element ratios in leaf stem and root of different species Table S1 The difference of stoichiometric traits of C N and P concentrations and their element ratios in different organs of C gracilis between epi-

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic andterrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at httpswwwmdpicom1999-490712116s1 Figure S1 The mean stoichiometric homeostasis (H) values for N and Pelements and their element ratios in leaf stem and root of different species Table S1 The differenceof stoichiometric traits of C N and P concentrations and their element ratios in different organs of C

Forests 2021 12 16 10 of 15

4 Discussion41 Differences in Nutrient Use Strategy between Epiphytic and Terrestrial Individuals ofFacultative Epiphytes

In our field experiment the concentrations of N and P in epiphytic individuals ofthree species in the natural forest were higher than those in terrestrial individuals but CNCP and NP ratios in the former were lower than the latter (Tables S1ndashS3) This agreedwith the results from fern in our study area and bromeliads in humid montane forest andlowland forests elsewhere [134748] CN and CP ratios reflect the carbon fixation per unitnutrient the higher N and P concentration and lower CN and CP values are associatedwith luxury consumption of plants [164950] Accordingly in our study the use of N andP by epiphytic individuals were more luxurious than terrestrial individuals which agreeswith the result that terrestrial individuals of fern are more efficient in nutrient use thanepiphytic individuals [13] The plasticity in nutrient use strategy of facultative epiphytes isconsidered to be an adaptive mechanism to different environmental conditions in orderto exploit the available resources efficiently [1213] Our results indicated that facultativeepiphytes had more flexible plasticity in nutrient use strategies to adapt habitat shifts

Winkler and Zotz [51] found that epiphytes possessed lower nutrient use efficiencythan that of terrestrial plants Epiphytes are generally considered to survive in the nutrientintermittent epiphytic habitats through luxurious consumption of nutrients [12] Theyusually absorb large amounts of nutrients and store them in vacuoles when the nutrient isabundant [142949] Accordingly the lower N and P use of epiphytic individuals may berelated to the luxury uptake of N and P in epiphytic habitats Furthermore we found thatvariation in the elemental composition of epiphytic individuals exceeded that of terrestrialindividuals most likely because epiphytic habitats are more heterogeneous [552] and theelemental composition of plants is influenced by other environmental factors (eg wateravailability light intensity) besides soil alone [3849]

42 Effects of Nutrient Limitation on the Distribution of Three Facultative Epiphytes

The foliar NP ratio can be used as an indicator of the type of nutrient limitationon plant growth the lower NP ratios indicate the plants were limited by N [244953]whereas the higher NP ratios indicates P limitations were present [2954] Epiphytes areusually restricted by P [55] but in our research nutrient restriction types differ amongthe three species The NP ratio of terrestrial and epiphytic individuals of E monandrumand epiphytic individuals of B longifolia was lower than 12 (p-values = 0214 0526 0225respectively) (Tables S2 and S3) suggesting the growth of which was restricted by Navailability [29] Since the NP ratio in terrestrial and epiphytic individuals of C gracilisand terrestrial individuals of B longifolia was higher than 12 (p-values = 0004 0227 0009respectively) yet still lower than 16 (p-values = 0 005 0 0006 respectively) (Tables S1 andS3) we interpreted this to mean that the growth of which were limited by N or co-limited byN and P [28] Our results were consistent with the finding that facultative epiphytes on datepalms (Phoenix dactylifera) in southeastern Spain were subject to N-limited conditions [14]Furthermore in our research the lower NP ratio in epiphytic than terrestrial individualsimplied that N-restriction in the epiphytic habitat was more pronounced The higher Pconcentration in epiphytic individuals probably increases the demands for N thus makingthem more pronounced Furthermore the higher P concentration in plants can alleviate thedamage caused by drought stress [49] In this study the higher P concentration in epiphyticindividuals may reflect their adaptation to epiphytic habitats where water is intermittent

The factors that drive the distribution of facultative epiphytes are not clear butprevious results suggested that environmental factors can affect the distribution of epi-phytes [1446] For the three species in our research most individuals of E monandrum andB longifolia grow on the bark with mass mat or thick humus while the majority of C gracilisgrow on the forest floor For E monandrum and B longifolia the N-restriction in epiphytichabitats was stronger than terrestrial habitats therefore they may gradually shift to forestfloor to alleviate N limitation For C gracilis the type of nutrient limitation in terrestrial and

Forests 2021 12 16 11 of 15

epiphytic habitats was same this indicated that the nutrient status in epiphytic habitatscan satisfy its requirement Previous research indicated that the interspecific competition interrestrial habitats was more intense than that in epiphytic habitats [7] Therefore C gracilismay shift to epiphytic habitats to escape interspecific competition

43 Differences in Stoichiometric Homeostasis among Species Organs and Elements ofFacultative Epiphytes

Traditional theory predicts that the stoichiometric homeostasis of autotroph was rela-tively weak [1756] However mounting data suggest that plants have a strong regulatorycapacity for their element composition [202237] Our results of the field and greenhousecultivation experiment indicated that the values of HN HP and HNP of leaves were allgt4 (p = 0002 0004 respectively) (Figure 4 Table S4) both showed strong homeostasiswhich did not support our second hypothesis that the homeostasis of facultative epiphytesis weak These results for facultative epiphytes in our study are also at odds with previousresearch concluded that a weak homeostasis of plants implied higher ecological adapt-ability and better suitability to a more variable environment [33] This discrepancy maybe because that conclusion about relationship between stoichiometric homeostasis andecological adaptability of plants was almost based on studies of plant leaves [3349] Theelemental composition of leaves is insensitive to nutrient changes in the soil instead rootsare more valid indicators of nutrient change in the environment [203857ndash59] Therefore aleafrsquos stoichiometric homeostasis cannot accurately reflect the ecological adaptability ofplants [3860] In our work here although leaves did have strong stoichiometric home-ostasis it was rather weak for roots (Table S4) The main function of roots is to absorbmineral nutrients and water from the soil [33] their plastic nutrient absorption is helpfulfor facultative epiphytes to efficiently exploit the available resources in different habitatsThis result indicated that facultative epiphytes could adapt to variable environment bychanging the elemental composition of their roots

Moreover the difference in stoichiometric homeostasis among the three species in ourfield study was not significant and the stoichiometric homeostasis mainly affected by or-gans (Table S5) likely because all three share a similar species richness in the sampling areaand their ecological strategy and adaptability to the environment were comparable [22]This was also confirmed by the consistent response of the two species to nutrient additionin the greenhouse cultivation experiments in our study (Table 3)

Different organs of the same species exhibit disparate stoichiometric characteristicsto better adapt to the environment [162033] We found that stoichiometric homeostasiswas stronger in aboveground than belowground of the three facultative epiphytes Rootswere more sensitive to changes of elements concentrations in environmental resourceswhich agrees with findings reported for terrestrial grass and woody plants [21223138]However unlike the related studies on terrestrial woody plants [38] the stems of C gracilisand E monandrum show strong stoichiometric homeostasis (Table S4) This may be relatedto the insensitivity of some key elements in stems of facultative epiphyte to changes insoil nutrient concentrations The stems of woody and herbaceous plants take differentfunctions the stems of woody plants mainly play transportation and supporting rolewhile the stems of herbaceous plants have less structural C than woody plants yet bearthe functions of photosynthesis transportation and the supporting action [20] Thereforethe stems of herbaceous plants need relatively stable supply of elements to maintain theirphysiological functions

The strength of homeostasis is also related to the elemental concentration [61] Previ-ous research for terrestrial plants indicated that the homeostasis of macro-elements washigher than trace elements [3461] In our study the value of stoichiometric homeostasisof N and P in the three facultative epiphyte species showed the order of HN gt HP This isconsistent with results from vertebrates and trees with greater control over high-demandelements [34ndash36] Therefore the facultative epiphytic plants may also have greater con-trol over macro-elements highly demanded for physiology functioning However ourresearch only focused on the stoichiometric homeostasis of N and P elements in facultative

Forests 2021 12 16 12 of 15

epiphytes the stoichiometric homeostasis of trace elements remains unknown Thereforewhether the stoichiometric homeostasis of macro-elements in facultative epiphytes is higherthan trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumulationof N within a given plant tissue is usually accompanied by an increase in its P content thehomeostasis of NP is stronger than N and P elements [1762] However in our study thestoichiometric homeostasis value of NP in leaf and root were all higher than that of P butlower than that of N (Figure 3) Based on the theory that limiting elements should havestrong homeostasis [313463] we speculate that facultative epiphytes may be restricted byN availability These plants therefore would take strong regulation of N to cope with theunpredictable fluctuations of N elements in their habitat

5 Conclusions

This study systematically elucidates the nutrient use and ecological adaptation strate-gies of facultative epiphytes from the perspective of stoichiometry using field investigationand greenhouse cultivation experiments Our study showed that epiphytic individualsof facultative epiphytes survive intermittent habitat via luxury consumption of nutrientswhile terrestrial individuals use more conservative nutrients (Figure 5) The nutrient usestrategies of facultative epiphytes were flexible which enabled facultative epiphytes toexploit bark and soil interchangeably Facultative epiphytes had strong control over N andP concentrations and NP ratio of leaves and stems while roots had weaker stoichiometricstability These findings suggested that to adapt to the variable habitats in montane forestecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulatingthe absorption and composition of elements in roots

|

Forests 2021 12 x FOR PEER REVIEW 12 of 15

trol over macro-elements highly demanded for physiology functioning However our research only focused on the stoichiometric homeostasis of N and P elements in faculta-tive epiphytes the stoichiometric homeostasis of trace elements remains unknown Therefore whether the stoichiometric homeostasis of macro-elements in facultative epi-phytes is higher than trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumu-lation of N within a given plant tissue is usually accompanied by an increase in its P content the homeostasis of NP is stronger than N and P elements [1762] However in our study the stoichiometric homeostasis value of NP in leaf and root were all higher than that of P but lower than that of N (Figure 3) Based on the theory that limiting ele-ments should have strong homeostasis [313463] we speculate that facultative epiphytes may be restricted by N availability These plants therefore would take strong regulation of N to cope with the unpredictable fluctuations of N elements in their habitat

5 Conclusions This study systematically elucidates the nutrient use and ecological adaptation

strategies of facultative epiphytes from the perspective of stoichiometry using field in-vestigation and greenhouse cultivation experiments Our study showed that epiphytic individuals of facultative epiphytes survive intermittent habitat via luxury consumption of nutrients while terrestrial individuals use more conservative nutrients (Figure 5) The nutrient use strategies of facultative epiphytes were flexible which enabled facultative epiphytes to exploit bark and soil interchangeably Facultative epiphytes had strong control over N and P concentrations and NP ratio of leaves and stems while roots had weaker stoichiometric stability These findings suggested that to adapt to the variable habitats in montane forest ecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulating the absorption and composition of elements in roots

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic and terrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at wwwmdpicomxxxs1 Fig-ure S1 The mean stoichiometric homeostasis (H) values for N and P elements and their element ratios in leaf stem and root of different species Table S1 The difference of stoichiometric traits of C N and P concentrations and their element ratios in different organs of C gracilis between epi-

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic andterrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at httpswwwmdpicom1999-490712116s1 Figure S1 The mean stoichiometric homeostasis (H) values for N and Pelements and their element ratios in leaf stem and root of different species Table S1 The differenceof stoichiometric traits of C N and P concentrations and their element ratios in different organs of C

Forests 2021 12 16 11 of 15

epiphytic habitats was same this indicated that the nutrient status in epiphytic habitatscan satisfy its requirement Previous research indicated that the interspecific competition interrestrial habitats was more intense than that in epiphytic habitats [7] Therefore C gracilismay shift to epiphytic habitats to escape interspecific competition

43 Differences in Stoichiometric Homeostasis among Species Organs and Elements ofFacultative Epiphytes

Traditional theory predicts that the stoichiometric homeostasis of autotroph was rela-tively weak [1756] However mounting data suggest that plants have a strong regulatorycapacity for their element composition [202237] Our results of the field and greenhousecultivation experiment indicated that the values of HN HP and HNP of leaves were allgt4 (p = 0002 0004 respectively) (Figure 4 Table S4) both showed strong homeostasiswhich did not support our second hypothesis that the homeostasis of facultative epiphytesis weak These results for facultative epiphytes in our study are also at odds with previousresearch concluded that a weak homeostasis of plants implied higher ecological adapt-ability and better suitability to a more variable environment [33] This discrepancy maybe because that conclusion about relationship between stoichiometric homeostasis andecological adaptability of plants was almost based on studies of plant leaves [3349] Theelemental composition of leaves is insensitive to nutrient changes in the soil instead rootsare more valid indicators of nutrient change in the environment [203857ndash59] Therefore aleafrsquos stoichiometric homeostasis cannot accurately reflect the ecological adaptability ofplants [3860] In our work here although leaves did have strong stoichiometric home-ostasis it was rather weak for roots (Table S4) The main function of roots is to absorbmineral nutrients and water from the soil [33] their plastic nutrient absorption is helpfulfor facultative epiphytes to efficiently exploit the available resources in different habitatsThis result indicated that facultative epiphytes could adapt to variable environment bychanging the elemental composition of their roots

Moreover the difference in stoichiometric homeostasis among the three species in ourfield study was not significant and the stoichiometric homeostasis mainly affected by or-gans (Table S5) likely because all three share a similar species richness in the sampling areaand their ecological strategy and adaptability to the environment were comparable [22]This was also confirmed by the consistent response of the two species to nutrient additionin the greenhouse cultivation experiments in our study (Table 3)

Different organs of the same species exhibit disparate stoichiometric characteristicsto better adapt to the environment [162033] We found that stoichiometric homeostasiswas stronger in aboveground than belowground of the three facultative epiphytes Rootswere more sensitive to changes of elements concentrations in environmental resourceswhich agrees with findings reported for terrestrial grass and woody plants [21223138]However unlike the related studies on terrestrial woody plants [38] the stems of C gracilisand E monandrum show strong stoichiometric homeostasis (Table S4) This may be relatedto the insensitivity of some key elements in stems of facultative epiphyte to changes insoil nutrient concentrations The stems of woody and herbaceous plants take differentfunctions the stems of woody plants mainly play transportation and supporting rolewhile the stems of herbaceous plants have less structural C than woody plants yet bearthe functions of photosynthesis transportation and the supporting action [20] Thereforethe stems of herbaceous plants need relatively stable supply of elements to maintain theirphysiological functions

The strength of homeostasis is also related to the elemental concentration [61] Previ-ous research for terrestrial plants indicated that the homeostasis of macro-elements washigher than trace elements [3461] In our study the value of stoichiometric homeostasisof N and P in the three facultative epiphyte species showed the order of HN gt HP This isconsistent with results from vertebrates and trees with greater control over high-demandelements [34ndash36] Therefore the facultative epiphytic plants may also have greater con-trol over macro-elements highly demanded for physiology functioning However ourresearch only focused on the stoichiometric homeostasis of N and P elements in facultative

Forests 2021 12 16 12 of 15

epiphytes the stoichiometric homeostasis of trace elements remains unknown Thereforewhether the stoichiometric homeostasis of macro-elements in facultative epiphytes is higherthan trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumulationof N within a given plant tissue is usually accompanied by an increase in its P content thehomeostasis of NP is stronger than N and P elements [1762] However in our study thestoichiometric homeostasis value of NP in leaf and root were all higher than that of P butlower than that of N (Figure 3) Based on the theory that limiting elements should havestrong homeostasis [313463] we speculate that facultative epiphytes may be restricted byN availability These plants therefore would take strong regulation of N to cope with theunpredictable fluctuations of N elements in their habitat

5 Conclusions

This study systematically elucidates the nutrient use and ecological adaptation strate-gies of facultative epiphytes from the perspective of stoichiometry using field investigationand greenhouse cultivation experiments Our study showed that epiphytic individualsof facultative epiphytes survive intermittent habitat via luxury consumption of nutrientswhile terrestrial individuals use more conservative nutrients (Figure 5) The nutrient usestrategies of facultative epiphytes were flexible which enabled facultative epiphytes toexploit bark and soil interchangeably Facultative epiphytes had strong control over N andP concentrations and NP ratio of leaves and stems while roots had weaker stoichiometricstability These findings suggested that to adapt to the variable habitats in montane forestecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulatingthe absorption and composition of elements in roots

|

Forests 2021 12 x FOR PEER REVIEW 12 of 15

trol over macro-elements highly demanded for physiology functioning However our research only focused on the stoichiometric homeostasis of N and P elements in faculta-tive epiphytes the stoichiometric homeostasis of trace elements remains unknown Therefore whether the stoichiometric homeostasis of macro-elements in facultative epi-phytes is higher than trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumu-lation of N within a given plant tissue is usually accompanied by an increase in its P content the homeostasis of NP is stronger than N and P elements [1762] However in our study the stoichiometric homeostasis value of NP in leaf and root were all higher than that of P but lower than that of N (Figure 3) Based on the theory that limiting ele-ments should have strong homeostasis [313463] we speculate that facultative epiphytes may be restricted by N availability These plants therefore would take strong regulation of N to cope with the unpredictable fluctuations of N elements in their habitat

5 Conclusions This study systematically elucidates the nutrient use and ecological adaptation

strategies of facultative epiphytes from the perspective of stoichiometry using field in-vestigation and greenhouse cultivation experiments Our study showed that epiphytic individuals of facultative epiphytes survive intermittent habitat via luxury consumption of nutrients while terrestrial individuals use more conservative nutrients (Figure 5) The nutrient use strategies of facultative epiphytes were flexible which enabled facultative epiphytes to exploit bark and soil interchangeably Facultative epiphytes had strong control over N and P concentrations and NP ratio of leaves and stems while roots had weaker stoichiometric stability These findings suggested that to adapt to the variable habitats in montane forest ecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulating the absorption and composition of elements in roots

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic and terrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at wwwmdpicomxxxs1 Fig-ure S1 The mean stoichiometric homeostasis (H) values for N and P elements and their element ratios in leaf stem and root of different species Table S1 The difference of stoichiometric traits of C N and P concentrations and their element ratios in different organs of C gracilis between epi-

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic andterrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at httpswwwmdpicom1999-490712116s1 Figure S1 The mean stoichiometric homeostasis (H) values for N and Pelements and their element ratios in leaf stem and root of different species Table S1 The differenceof stoichiometric traits of C N and P concentrations and their element ratios in different organs of C

Forests 2021 12 16 12 of 15

epiphytes the stoichiometric homeostasis of trace elements remains unknown Thereforewhether the stoichiometric homeostasis of macro-elements in facultative epiphytes is higherthan trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumulationof N within a given plant tissue is usually accompanied by an increase in its P content thehomeostasis of NP is stronger than N and P elements [1762] However in our study thestoichiometric homeostasis value of NP in leaf and root were all higher than that of P butlower than that of N (Figure 3) Based on the theory that limiting elements should havestrong homeostasis [313463] we speculate that facultative epiphytes may be restricted byN availability These plants therefore would take strong regulation of N to cope with theunpredictable fluctuations of N elements in their habitat

5 Conclusions

This study systematically elucidates the nutrient use and ecological adaptation strate-gies of facultative epiphytes from the perspective of stoichiometry using field investigationand greenhouse cultivation experiments Our study showed that epiphytic individualsof facultative epiphytes survive intermittent habitat via luxury consumption of nutrientswhile terrestrial individuals use more conservative nutrients (Figure 5) The nutrient usestrategies of facultative epiphytes were flexible which enabled facultative epiphytes toexploit bark and soil interchangeably Facultative epiphytes had strong control over N andP concentrations and NP ratio of leaves and stems while roots had weaker stoichiometricstability These findings suggested that to adapt to the variable habitats in montane forestecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulatingthe absorption and composition of elements in roots

|

Forests 2021 12 x FOR PEER REVIEW 12 of 15

trol over macro-elements highly demanded for physiology functioning However our research only focused on the stoichiometric homeostasis of N and P elements in faculta-tive epiphytes the stoichiometric homeostasis of trace elements remains unknown Therefore whether the stoichiometric homeostasis of macro-elements in facultative epi-phytes is higher than trace elements still requires further testing and verification

Due to the synergistic changes of N and P within plant tissues in that the accumu-lation of N within a given plant tissue is usually accompanied by an increase in its P content the homeostasis of NP is stronger than N and P elements [1762] However in our study the stoichiometric homeostasis value of NP in leaf and root were all higher than that of P but lower than that of N (Figure 3) Based on the theory that limiting ele-ments should have strong homeostasis [313463] we speculate that facultative epiphytes may be restricted by N availability These plants therefore would take strong regulation of N to cope with the unpredictable fluctuations of N elements in their habitat

5 Conclusions This study systematically elucidates the nutrient use and ecological adaptation

strategies of facultative epiphytes from the perspective of stoichiometry using field in-vestigation and greenhouse cultivation experiments Our study showed that epiphytic individuals of facultative epiphytes survive intermittent habitat via luxury consumption of nutrients while terrestrial individuals use more conservative nutrients (Figure 5) The nutrient use strategies of facultative epiphytes were flexible which enabled facultative epiphytes to exploit bark and soil interchangeably Facultative epiphytes had strong control over N and P concentrations and NP ratio of leaves and stems while roots had weaker stoichiometric stability These findings suggested that to adapt to the variable habitats in montane forest ecosystem facultative epiphytes maintain stable metabolic activities of leaves by regulating the absorption and composition of elements in roots

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic and terrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at wwwmdpicomxxxs1 Fig-ure S1 The mean stoichiometric homeostasis (H) values for N and P elements and their element ratios in leaf stem and root of different species Table S1 The difference of stoichiometric traits of C N and P concentrations and their element ratios in different organs of C gracilis between epi-

Figure 5 Summary of links between nutrient composition and nutrient use strategy of epiphytic andterrestrial individuals of facultative epiphytes The (+) denotes that effects are positive

Supplementary Materials Supplementary materials can be found at httpswwwmdpicom1999-490712116s1 Figure S1 The mean stoichiometric homeostasis (H) values for N and Pelements and their element ratios in leaf stem and root of different species Table S1 The differenceof stoichiometric traits of C N and P concentrations and their element ratios in different organs of C

Forests 2021 12 16 13 of 15

gracilis between epiphytic and terrestrial habitats Table S2 The difference of stoichiometric traitsof C N and P concentrations and their element ratios in different organs of E monandrum betweenepiphytic and terrestrial habitats Table S3 The difference of stoichiometric traits of C N and Pconcentrations and their element ratios in different organs of B longifolia between epiphytic andterrestrial habitats Table S4 The stoichiometric homeostasis values for different elements organsand species in the field experiment Table S5 Comparison of stoichiometric homeostasis values(mean plusmn SE) for different organs and species in the field study and the effects of organ species andtheir interactions on stoichiometry homeostasis

Author Contributions TZ and WL conceived and designed the study TZ conducted the culti-vation experiment TZ TH and DT conducted the field experiment TZ collected and analyzeddata TZ WL YM and YW wrote the manuscript All authors contributed critically to the draftsand gave final approval for publication All authors have read and agreed to the published versionof the manuscript

Funding This study was funded by the National Natural Science Foundation of China (3177049642071071 41471050) the Biodiversity Conservation Strategy Program of Chinese Academy of Sciences(ZSSD-016) and the ldquoCAS 135 Programrdquo (2017XTBG-T01)

Institutional Review Board Statement Not applicable

Informed Consent Statement Not applicable

Data Availability Statement The data used in this study are available on request from the corre-sponding author

Acknowledgments We thank the staff of the Ailaoshan Subtropical Forest Ecosystem ResearchStation for their assistance during the field sampling and the Biogeochemical Laboratory of Xishuang-banna Tropical Botanical Garden for helping us analyze the elemental contents of plant samplesFurther we would like to thank Richard T Corlett for his constructive suggestions in the structureand presentation of this paper

Conflicts of Interest The authors declare no conflict of interest

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University of California Press Oakland CA USA 20123 Zotz G Winkler U Aerial roots of epiphytic orchids The velamen radicum and its role in water and nutrient uptake Oecologia

2013 171 733ndash741 [CrossRef]4 Benzing DH Vascular Epiphytes General Biology and Related Biota Cambridge University Press New York NY USA 19905 Lu HZ Liu WY Yu FH Song L Xu XL Wu CS Zheng YL Li YP Gong HD Chen K Higher clonal integration in

the facultative epiphytic fern Selliguea griffithiana growing in the forest canopy compared with the forest understory Ann Bot2015 116 113ndash122 [CrossRef] [PubMed]

6 Zotz G lsquoHemiepiphytersquo A confusing term and its history Ann Bot 2013 111 1015ndash1020 [CrossRef] [PubMed]7 Lu HZ Brooker R Song L Liu WY Sack L Zhang JL Yu FH When facilitation meets clonal integration in forest

canopies New Phytol 2020 225 135ndash142 [CrossRef] [PubMed]8 Li S Liu WY Li DW Song L Chen K Fu Y Slower rates of litter decomposition of dominant epiphytes in the canopy than

on the forest floor in a subtropical montane forest southwest China Soil Biol Biochem 2014 70 211ndash220 [CrossRef]9 Theacutery M Forest light and its influence on habitat selection Plant Ecol 2001 153 251ndash261 [CrossRef]10 Zotz G Hietz P The physiological ecology of vascular epiphytes Current knowledge open questions J Exp Bot 2001 52

2067 [CrossRef]11 Zhang Q Chen JW Li BG Cao KF The effect of drought on photosynthesis in two epiphytic and two terrestrial tropical fern

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2000 30 1ndash6724 Guumlsewell S N P ratios in terrestrial plants Variation and functional significance New Phytol 2004 164 243ndash266 [CrossRef]25 Guumlsewell S Responses of wetland graminoids to the relative supply of nitrogen and phosphorus Plant Ecol 2005 176 35ndash55

[CrossRef]26 Peaeliguelas J Poulter B Sardans J Ciais P van der Velde M Bopp L Boucher O Godderis Y Hinsinger P Llusia J et al

Human-induced nitrogenndashphosphorus imbalances alter natural and managed ecosystems across the globe Nat Commun 2013 43934

27 Ma W Li J Jimoh SO Zhang Y Guo F Ding Y Li X Hou X Stoichiometric ratios support plant adaption to grazingmoderated by soil nutrients and root enzymes PeerJ 2019 7 e7047 [CrossRef] [PubMed]

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temporal stability and responses to global change Ecology 2015 96 2328ndash2335 [CrossRef]32 Dijkstra FA Pendall E Morgan JA Blumenthal DM Carrillo Y Lecain DR Follett RF Williams DG Climate change

alters stoichiometry of phosphorus and nitrogen in a semiarid grassland New Phytol 2012 196 807ndash815 [CrossRef]33 Persson J Fink P Goto A Hood JM Jonas J Kato S To be or not to be what you eat Regulation of stoichiometric

homeostasis among autotrophs and heterotrophs Oikos 2010 119 741ndash751 [CrossRef]34 Han WX Fang JY Reich PB Ian WF Wang ZH Biogeography and variability of eleven mineral elements in plant leaves

across gradients of climate soil and plant functional type in China Ecol Lett 2011 14 788ndash796 [CrossRef]35 Karimi R Folt CL Beyond macronutrients Element variability and multielement stoichiometry in freshwater invertebrates

Ecol Lett 2006 9 1273ndash1283 [CrossRef] [PubMed]36 Tang ZY Xu WT Zhou GY Bai YF Li JX Tang XL Chen DM Liu Q Ma WH Xiong GM et al Patterns of plant

carbon nitrogen and phosphorus concentration in relation to productivity in Chinarsquos terrestrial ecosystems Proc Natl Acad SciUSA 2018 115 4033ndash4038 [CrossRef] [PubMed]

37 Jiang LL Zeng CS Shao JJ Zhou XH Plant nutrient dynamics and stoichiometric homeostasis of invasive species Spartinaalterniflora and native Cyperus malaccensis var brevifolius in the Minjiang River estuarine wetlands Chin J Plant Ecol 2017 41405ndash460

38 Schreeg LA Santiago LS Wright SJ Turner BL Stem root and older leaf NP ratios are more responsive indicators of soilnutrient availability than new foliage Ecology 2014 95 2062ndash2068 [CrossRef] [PubMed]

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40 Liu J Fang X Tang X Wang W Zhou G Xu S Huang W Wang G Yan J Ma K et al Patterns and controlling factorsof plant nitrogen and phosphorus stoichiometry across Chinarsquos forests Biogeochemistry 2019 143 191ndash205 [CrossRef]

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Regrowth Stands in Central-West Yunnan Province China Vegetatio 1995 119 1ndash13

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45 Li S Liu W Wang L Ma W Song L Biomass diversity and composition of epiphytic macrolichens in primary and secondaryforests in the subtropical Ailao Mountains SW China Forest Ecol Manag 2011 261 1760ndash1770 [CrossRef]

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