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Abiotic factors modulate phenotypic plasticityin an apomictic shrub [Miconia albicans (SW.) Triana]along a soil fertility gradient in a Neotropical savanna

Cibele S. BedettiA,B, Dbora B. AguiarA, Maria C. JannuzziA, Maria Z. D. MouraA

and Fernando A. O. SilveiraA,B,C

AFaculdade de Cincias e da Sade, Centro Universitrio UNA, Campus Guajajaras, Belo Horizonte,

Minas Gerais, Brazil.BDepartamento de Biologia Vegetal, Instituto de Cincias Biolgicas, Universidade Federal

de Minas Gerais, Avenida Antnio Carlos 6627, Pampulha, Belo Horizonte, MG 31270-901, Brazil.CCorresponding author. Email: faosilveira@gmail.com

Abstract. Phenotypic plasticity is an important means by which plants cope with environmental heterogeneity; therefore,understanding variation in plant traits in heterogeneous habitats is important to predict responses to changing environments.In this study, we examined the patterns of intraspecific variation in leaf traits of Miconia albicans (Melastomataceae),awidespread,obligatoryapomicticshrub,acrossasoilfertilitygradientintheCerrado(Braziliansavanna).Wepredictedhighplasticity because selection favours high phenotypic plasticity in asexual populations with low genetic variability. Leavesweresampledin camposujo (grassland), cerrado (savanna)and cerrado(woodland)in south-easternBrazil during bothdryand rainy seasons to calculate leaf area, specific leaf area, leaf tissue thickness, trichome and stomata density. We foundsignificantbetween-season variationin leaftraits, indicating thatthe productionof season-specificleavesisastrategytocopewith the strong seasonality. Both multivariate analysis and the relative distance plasticity index indicate lower plasticityduringthe dryseason, especially under shade.Our results show that thephenotypicplasticitycan be modulated by changes inabiotic factors and the combination of shade and drought can limit the expression of phenotypic plasticity.

Introduction

Changes in leaf structure and function following changes inenvironmental factors are widely documented worldwide.Variation in leaf traits can play an important role in theperformance of species across a broad range of environmentalgradients or highly heterogeneous habitats (Sandquist andEhleringer 1997; Valladares et al. 2006, 2007). By producingnew and structurally altered leaves, plants can respond to changesin irradiance, light spectral quality, CO2 concentration, soilfertility and moisture. Environmental properties are known toaffect leaf morphology, anatomy and, ultimately, physiology(Lee et al. 2000; Marques et al. 2000; Lake et al. 2001;

Nicotra et al. 2007; Chen et al. 2010; Sanches et al. 2010).Due to a strong linkage between structure and function, plantsin heterogeneous habitats produce morphologically adjustedleaves that optimise carbon acquisition and water economy,which may result in increased fitness. Phenotypic plasticity(lato sensu) is an important means by which plants cope withenvironmental heterogeneity (Valladares et al. 2006, 2007), acommon characteristic in the Brazilian Cerrado.

The Cerrado sensu lato (a Neotropical savanna) originallycovered 2 million km2, extending from the southern borders ofthe Amazon forest to outlying areas in the southern portions ofBrazil, occupying an altitudinal range from near sea level to

1.800 m (Oliveira-Filho and Ratter 2002). The Cerradodeveloped on old, highly weathered, deep or shallow soils,most of which are dystrophic, with low pH and high levels ofexchangeable aluminium (Ratter et al. 1997). The climate isseasonal with a dry and cold season from April to Septemberfollowed by a 6-month rainy and hot season (Oliveira-Filho andRatter2002). Duringthe drywintersthe high evaporativedemandleads to strong stomatal control of transpiration, affecting thepatterns of gas exchange and photosynthesis (Scholz et al.2008). Plants adapted to seasonal savannas have evolvedmechanisms that maximise water economy during this period.Adaptations include ecological, physiological, anatomical, and

chemical mechanisms such as deciduousness, strong stomatalcontrol and the production of leaves with dense trichome cover,thick cuticles, hipostomatic leaves, well developed palisadetissue, sunken stomata, thickened and flat epidermal cells andidioblasts bearing crystals and phenolic compounds (Franco2002; Reis et al. 2005; Scholz et al. 2008; Bieras and Sajo2009; Rossatto and Kolb 2009). Many of these traits areregarded as protection against herbivores (Marquis et al. 2001,2002; Bieras and Sajo 2009).

Spatial heterogeneity in the Cerrado landscape is largelymaintained by soil fertility (Oliveira-Filho and Ratter 2002).The landscape consists of cerrado on the well drained

CSIRO PUBLISHING

www.publish.csiro.au/journals/ajb Australian Journal of Botany, 2011, 59, 274282

CSIRO 2011 10.1071/BT10275 0067-1924/11/030274

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interfluves with riparian forests bordering watercourses. Thephytophysiognomies range from dense grassland to woodlands(Ratteret al. 1997; Oliveira-Filho and Ratter2002) with a greatvariation in the proportion of the herbaceous layer (made upof grasses, sedges and subshrubs) and the woody communities,formed by relatively small, gnarled and scattered tree and

shrubs (Furley 1999). Since these contrasting environmentsprovide different selective pressures, one would expectdisruptive selection to generate a dichotomic intraspecificvariation pattern in traits associated with habitat selection(Lemos-Filho et al. 2008). Competition for water is moreintense in open phytophysiognomies whereas competition forlight is reported in shaded ones. The outcome of this dichotomyis that plants growing in open phytophysiognomies produceleaves with well developed cuticles, increased trichomedensity and mesophyll thickness. Plants at woodland savannas,on the other hand, produce leaves that maximise light capture,including increased leaf area, lower specific leaf area (SLA),high chlorophyll content and reduced stomata size and pore(Marques et al . 2000; Valladares and Niinemets 2008;

Rossatto et al. 2009).Variations in leaf structure should ultimately lead to

differential water relations and light capture mechanismsbetween shade and sun leaves. Thus, a better understanding onthe variability of leaf traits in vegetation mosaics can enhanceour knowledge on plant functional ecology in a changing world.In spite of the documented changes in plant architecture, leafspecific area, leaf anatomy in contrasting habitats, we do notfully appreciate how plant traits vary along ecological gradients.So far, changes in leaf traits in contrasting habitats have beeninterpreted as the outcomes of selective pressures operating atopposite directions, but grasslands and woodlands of Cerradomay represent the extremes of a continuum of variation rather

than a product of a simple dichotomy.By means of quantitative leaf morpho-anatomy of individualsgrowing along a soil fertility gradient, we aimed to investigatechanges in leaf structure in Miconia albicans during both dryand rainy seasons. We examined variability of traits associatedwith light capture and water economy, important traits foracclimation to heterogeneous environments, especially those

under strong seasonality. The study species, M. albicans (SW.)Triana (Melastomataceae), is an evergreen obligatory apomicticshrub occurring across the phytophysiognomies of the Cerrado,and is commonly found in grasslands and woodlands in thecore area of the biome (Goldenberg and Shepherd 1998;Goldenberg 2004). Our goal was to test the following

hypothesis: (i) M. albicans presents high plasticity becauseselection favours high phenotypic plasticity in asexualpopulations with low genetic variability (Richards 1997);(ii) M. albicans presents season-specific leaf types; (iii) thereis an increase in water economy-related leaf traits towardscanopy openness and an increase in light capture-related leaftraits towards canopy closure.

Material and methods

Study site and species

This study wascarried outat theEstaco Ecolgicade Pirapitinga(EEP), Trs Marias, Minas Gerais, south-eastern Brazil. Thestudy site is a man-made island formed during the filling of

theTrs Mariasreservoir in 1965 (from 1820.090Sto1823.150Sand 4517.40W to 4520.510W), at an altitude of 606 m abovesea level. The climate is seasonal with markedly rainy (fromOctober to April) and dry (from May to September) seasons.The mean annual temperature in the study site ranges from 21to25C and the average annual precipitation is 1200 mm. Duringthe dry season the air relative humidity drops dramatically(Fig. 1). The primary vegetation of the EEP is represented bythe phytophysiognomies of campo sujo, cerrado sensu strictoand cerrado, all establishing on sandy, shallow, aluminium-rich and nutrient-poor soils (Goncalves-Alvim and Fernandes2001). The campo sujo is a nutrient-poor soil grassland with acontinuous herbaceous layer and scattered small-sized shrubs

and trees. The cerrado sensu stricto is characterised by 3

8-m-tall shrubs and trees with nearly 30% cover and considerablegrass cover. The upper extreme of the gradient, the cerrado, isan almost closed woodland with crown cover ranging from50 to 90%, with 812 m trees casting a considerable shade sothat the understorey is much reduced (Oliveira-Filho and Ratter2002). Within this framework, there is increasing water

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availability towards the cerrado and an increasing lightgradient towards the campo sujo. These vegetation provide anatural gradient of structural complexity (cerrado> cerrado>campos sujo) as well as soil nutritional stress (camposujo> cerrado> cerrado), thus offering a great opportunityfor testing intraspecific variation of leaf morpho-anatomy.

Miconia albicans is a widespread evergreen species in SouthAmerica occurring from south-eastern Brazil to the south ofMxico (Goldenberg 2004). It is a 3-m-tall shrub, aluminium-accumulator (Haridasan 1988), which flowers from August toOctober. Its bird-dispersed berries ripen at the onset of therainy season and last until January. Leaves are simple,opposite, and elliptic, with a pubescent whitish abaxialsurface. Leaves are continuously produced year-round so thatexpanding and mature leaves can be found in both dry andrainy seasons.

Miconia albicans is a pioneer species occurring alongroadsides, disturbed areas and forest edges. Reproductivebiology studies have shown that M. albicans is an obligatoryapomictic species. The whitish, small and scentless flowers

are not visited by bees and produce pollen with null viability.These results were confirmed in studies carried out in differentlocations and different years of collection (Renner 1989;Goldenberg and Shepherd 1998; FAO Silveira, pers. obs.).

Leaf area and specific area

We established a 500-m transect in each phytophysiognomywhere 15 individuals located within 2 m from either sidesof the transect were randomly chosen and marked. The studysite is heterogeneous and constitutes a mosaic in whichphytophysiognomies may be separated by few meters.Distance between transects was 200400 m from each other.Ten expanded and mature leaves per individual of M. albicans

were sampled at 3-month intervals. Samples were taken inSeptember and December 2007 and March and July 2008,corresponding respectively to the dry-rainy transition, mid-rainy season, rainy-dry transition and mid-dry season.

Leaves were scanned and digitised. Leaf area was calculatedwith ImageTool software and then dried at 70C for 48h forleaf dry mass determination (Rozendaal et al. 2006). SLA(cm2 mg1) was obtained by dividing leaf area by dry mass.

Anatomical analysis

Mature leaves (n = 5 from each phytophysiognomy) werecollected in September 2007 and March 2008 (correspondingto the dry and rainy seasons, respectively), andfixed with FAA50

(37% formalin, acetic acid and 50% ethanol, 1:1:18, v/v;Johansen 1940). Histological sections were prepared withfreehand transverse sections at leaf midrib and petiole. Theobtained sections were cleared in sodium hypochlorite 50%,washed three times in water and stained with astra blue andfuchsin or toluidine blue (Kraus and Arduin 1997). The slideswere mounted in Kaisers glycerin gelatin (Kraus and Arduin1997) and analysed under a light microscope with a connectedcamera. Measurements of total leaf thickness, epidermis,palisade and spongy parenchyma thickness were carried outin 10 sections for each season. For such measures we used the

software Motic Images 19992000 (Micro-Optic 2000)connected to a Motic digital camera (all measurements weretaken in an established field of 200).

For the analysis of leaf epidermis, 10 leaf fragments wereseparated in sodium hypochlorite until complete detachment ofthe epidermis. The fragments were washed with water and

stained with astra blue and basic fuchsin. Threefi

elds of eachfragment were analysed and the number of stomata, trichomesand epidermal cells per square millimetre were counted in apredetermined field (400) coupled to a camera lucida. Thenumber of stomata and trichomes were counted per squaremillimetre.

Histochemical analysis

Histochemical procedures were carried in order to detect themain metabolite classes present in M. albicans leaves. Testswere run on freehand transverse sections, by using Sudan IIIfor lipids (Jensen 1962), ferric chloride for phenolics (Gahan1984), DMACA for flavanols and proantocianidins (Feuchtet al. 1986) and Lugol reagent for starch (Gahan 1984).Control reactions were carried out for all histochemicalmethods (Jensen 1962), following the suggestions of therespective authors.

Statistical analyses

A two-way repeated-measures ANOVA (with physiognomyand season as main factors) was used to determine significantdifferences in leaf area and SLA among phytophysiognomiesand seasons. This analysis is appropriate because it accountsfor data dependency and also allows for exploringphytophysiognomies and season interactions. Leaf area andSLA data were log10-transformed to meet the assumptions ofparametric analysis. The Tukey test was used as a multiple

comparison post-hoc test (Zar1996).To determine statistically significant differences in leaf

histometrics among phytophysiognomies, we conducted aone-way ANOVA when data were normally distributedand had homocedasticity. The Tukey test was used as amultiple comparison post-hoc test. Whenever necessary, log10-transformation was used to meet the assumptions of parametrictests. When data were not normally distributed and/or hadheterogeneous variances we used the non-parametric test ofKruskalWallis followed by the Conover test. To determinedifferences in histometric traits between rainy and dry seasonswe used the paired t-test for dependent samples.

A principal component analysis (PCA) was carried out

based on the correlation matrix of all histometric variables.The extent of variation of the anatomical traits related tophytophysiognomies was determined using a quantitativeestimate of phenotypic plasticity (lato sensu; Valladares et al.2006). The relative distance plasticity index (RDPI) measuresthe relative distances between the values of the chosenparameters measured for all pairs of individuals growing indifferent phytophysiognomies. These distances were calculatedaccording to the formula:

RDij!i0j0 dij!i0j0=xi0j0 xij 1

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where j and j0 are individuals of the different physiognomiesi a n d i0. RDPI varies from zero (no plasticity) to one(maximal plasticity) and was calculated according to thefollowing formula:

RDPI X

dij!i0j0 =xi0j0 xij=n 2

where n is the total of distances. RDPI data were submittedto ANOVA followed by Tukey test. For all analysis weestablished a value ofa=0.05.

Results

Leaf area and SLA

Phytophysiognomy and season had both significant effects onleaf area and SLA (Table 1). As expected, SLA and leaf areavalues followed the soil fertility gradient, with higher values forcerrado leaves, intermediate for cerrado leaves and lower incampo sujo leaves. A significant physiognomy by seasoninteraction was also observed for both variables

(Table 1, Fig. 2). This indicates that variation in leaf area andSLAacrossphytophysiognomiesisaffectedbyseason.TherewasnosignificantseasoneffectinbothleafareaandSLAintheshadedhabitat (Fig. 2).

Leaf anatomy and histometry

The leaf anatomy of M. albicans showed neither among-physiognomy nor between-season variation. The dermalsystem presents, in frontal view, ordinary cells with straightanticlinal walls on the adaxial surface (Fig. 3a) and slightlysinuous cell walls on the abaxial surface (Fig. 3b). Intransverse sections, a thick cuticle covers the adaxial surfacewhile numerous non-glandular branched trichomes occur onthe abaxial surface (Fig. 3c). The leaves are hypostomatic with

anomocytic stomata (Fig. 3b), which are slightly projected inrelation to the other epidermal cells (Fig. 3c). The mesophyllis dorsiventral, with a 1-layered palisade parenchyma and23-layered spongy parenchyma (Fig. 3c). In the mesophyll,just below the epidermis of the adaxial surface, we observedcrystal idioblasts containing druses (Fig. 3d). The xylem and

phloem present an arch-shaped collateral arrangement in thevascular system of the main vein in the middle leaf portion(Fig. 3e).

Thepetioleepidermisiscoveredbymanybranchedtrichomes,similar to those described for the leaf blade. The cortex andmedula are composed of parenchymatic cells with isodiametric

outline and isolated sclereids. The vascular system consists ofseveral arch-shaped collateral veins arranged around themedula (Fig. 3f). The histochemical tests detected the presenceof phenolic compounds in the cells of the chlorophyllparenchyma, in all phytophysiognomies for both seasons. Thehistochemical tests were negative for starch, lipids andflavonoids.

Except for cuticle thickness and trichome density, significantdifferences in most histometric traits were found between dryand rainy season leaves. This pattern held for the threephytophysiognomies clearly demonstrating the production ofseason-specific leaves. Higher means were found for rainyseason leaves compared with dry season leaves, but theinverse pattern was found for stomatal density (Table 2).

There were significant differences among the leaf histometrictraits among physiognomies. During the dry season, the leaftissue thickness showed low among-physiognomies variationwith statistical differences in campo sujo leaves comparedwith leaves from the cerrado and cerrado (Table 2).However, during the rainy season, the variation in allhistometric traits followed the ecological gradient, with lowervalues atcerrado, intermediate atcerrado and higher atcamposujo. For the spongy parenchyma thickness we did not find asignificant difference between campo sujo and cerrado, butcerrado leaves showed the lowest values. As a consequence

Table 1. Results of the two-way repeated-measures ANOVA for the

effects of physiognomy (P) and season (S) on the leaf area and specific

leaf area (SLA) of Miconia albicans in the Brazilian Cerrado.

Trait Source of variation

DF SS MS F-ratio Probability

Leaf area P 2 1.229 0.614 28.9

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Fig. 3. Miconia albicans leaf anatomy. (a) Frontal view of the adaxial epidermal surface showing ordinary cells with straight anticlinal walls (arrow).(b) Frontal view of the abaxial epidermal surface with non-glandular trichomes (black arrow) and stomata (white arrow). (c) Cross-section showing dorsiventralmesophyll with a thick cuticle covering the adaxial surface and numerous non-glandular branched trichomes on the abaxial surface (arrow). ( d) Presence ofcrystal idioblasts containing druse just below the epidermis of the adaxial surface. ( e) Transverse section of the main vein showing the collateral arrangementof xylem and phloem in the arched shape vascular system (dotted line). ( f) Transverse section of petiole where the cortex and pith are composed of

parenchymatic cells with few isolated sclereids (arrow) and the vascular system consists of various arched shape collateral veins. CT = cuticle; PP = palisadeparenchyma; SP = spongy parenchyma; AD = adaxial surface epidermis; CO = cortex; PI = pith.

Table 2. Means (s.d.) ofMiconia albicans leaf tissue thickness (mm) and trichome and stomatal density (number.mm2) in three phytophysiognomies

(CS, campo sujo; CE, cerrado; CD, cerrado) of the Brazilian Cerrado during the dry and rainy season

Dry season Rainy seasonAnatomical trait n CS CE CD F-ratio CS CE CD F-ratio

Adaxial cuticle 10 10.27 (1.17)Aa 7.27 (0.1)Ab 5.78 (1.23)Ac 38.71* 11.86 (2.3)Aa 9.41 (2.27)Bb 6.18 (0.86)Ac 21.75*

Adaxial epidermis 10 20.3 (2.14)A 18.0 (3.58)A 19.7 (5.08)A 1.04n.s. 24.07 (3.2)Ba 18.54 (2.71)Ab 12.62 (3.55)Bc 32.43*Palisade parenchyma 10 74.82 (5.60)Aa 44.3 (5.61)Ab 44.6 (11.80)Ab 45.79* 108.8 (20.3)Ba 72.3 (9.5)Bb 23.7 (3.7)Bc 24.35*Spongy parenchyma 10 43.2 (3.21)A 37.1 (5.21)A 40.2 (8.03)A 2.81n.s. 58.7 (11.7)Ba 57.8 (9.2)Ba 40.1 (5.9)Bb 11.17*

Total leaf 10 145.8 (9.07)Aa 110.7 (11.6)Ab 101.2 (25.2)Ab 19.4* 212 (29.6)Ba 155.2 (16.7)Bb 78.2 (25.7)Bc 131.7*Adaxial trichome

density30 0.011A 0.015A 0.011A 1.01n.s. 0.01A 0.012A 0.011A 0.39n.s.

Abaxial trichomedensity

30 0.11Aa 0.067Ab 0.058Ab 9.322* 0.076A 0.064A 0.062A 2.02n.s.

Stomata density 30 15.5 (4.3)Aa 9.4 (2.3)Ab 9.5 (2.1)Ab 39.9* 17.8 (3.2)Ba 8.03 (1.6)Bb 9(2.2)Ab 61.4*

Asterisk indicates a P-value

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of increased tissue thickness, total leaf thickness was greatest incampo sujo, followed by cerrado and, lastly, cerrado(Table 2). As expected, the trichome density was significantlyhigher in the abaxial surface of campo sujo leaves during thedry season (Table 2). The cerrado and cerrado showed similarvalues of stomatal density, which significantly differed from

those of the campo sujo. In both seasons, leaves from camposujo showed the higher stomatal density (Table 2).For the rainy season PCA, the principal component 1 (PC1)

separated the leaves from the three phytophysiognomies, inaccordance with the ecological gradient (Fig. 4a). The first twoaxes for the dry season PCA explained of 56.3% of total variancewhereas the first two axes for the rainy season PCA explained69.8% of total variance (Table 3). However, for the dry season,leaves from the shaded habitat (cerrado) were not separatedfrom those from the cerrado (Fig. 4b). For both seasons, mosthistometric traits were positively correlated with PC1. PC1 wasnot correlated with stomata density during the dry seasonand abaxial trichome density during the rainy season

(Table 3). Most variables were negatively correlated with PC2for both seasons. PC2 was not correlated with palisade thicknessduring the dry season and with epidermis and palisade thicknessduring the rainy season (Table 3).

Phenotypic plasticity (lato sensu) of leaf anatomical traitswashigher between campo sujo and cerrado than between cerrado

and cerrado as measured by the RDPI. This analysis alsoshowed higher RDPI values during the rainy season comparedwith the dry season for most cases, indicating between-seasonvariation in phenotypic plasticity (Table 4).

Discussion

This study documents a high level of leaf plasticity in awidespread, obligatory apomictic species (Goldenberg andShepherd 1998; Goldenberg 2004). Asexual populations tendto have low genetic variability. However, they have a very highproportion of loci fixed in the heterozygous phase, so selectionfavoured unusuallyhighlevels of phenotypic plasticity (Richards1997). Since M. albicans is subjected to differential selective

pressures throughout its broad geographic range, phenotypicplasticity may account for acclimation to heterogeneoushabitats, including the Cerrado and disturbed areas. High leafplasticityseems to be common inMiconia species (Marques etal.2000; Ely et al. 2005; Boergeret al. 2008) and is likely to play arole in adaptation to changing environments and contribute togeographic expansion.

More interesting, we have also demonstrated that abioticfactors can modulate the plastic response as evidence by lowerRDPI values at the dry season compared with the rainy season.Reduced plasticity at the dry season is also shown by the PCAdata. Thefact that plasticity observed in natureis often lower thanthat expected suggests the existence of costs and limits toplasticity (Valladares et al. 2007). The strong water deficit

during the dry season at the Cerrado poses strong limitationsto plant growth (Franco 2002) and drought can limit the plasticresponse during the dry season. Moreover, drought-shadeassociation is expected to be particularly costly (Valladareset al. 2007). The reduced leaf plasticity in M. albicansresulting from shade and drought interaction in the moreshaded habitat (cerrado) is in agreement with findings inother seasonal habitats (Valladares et al. 2007). Nevertheless,

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Fig. 4. Biplot representation of the trait scores on the first two axes ofthe principal component analysis (PCA) of histometric leaf traits ofMiconiaalbicans occurring in three Cerrado phytophysiognomies during rainy(a) and dry (b) seasons. Campo sujo (CS), cerrado sensu stricto (CE) andcerrado (CD).

Table 3. Principal components 1 (PC1) and 2 (PC2) obtained from

the matrix of correlation for leaf histometric trait of Miconia albicans in

three Cerrado phytophysiognomies

Histometric trait Dry season Rainy seasonPC1 PC2 PC1 PC2

Cuticle thickness 0.398 0.251 0.353 0.295Epidermis thickness 0.161 0.5601 0.378 0.03Palisade thickness 0.471 0.07083 0.433 0.07Spongy parenchyma thickness 0.242 0.4134 0.327 0.156

Total mesophyll thickness 0.397 0.3307 0.433 0.106Adaxial trichome density 0.202 0.4253 0.100 0.647Abaxial trichome density 0.318 0.3438 0.019 0.553Stomatal density 0.032 0.1315 0.31 0.12Percent palisade 0.372 0.1472 0.379 0.105Variance explained (%) 42.4 13.9 56.5 13.3

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we documented for the first time seasonal-dependent plasticlimitations.

We were also able to show the production of season-specific

leaf types in M. albicans. To our knowledge this is the secondreport on this strategy for a Neotropical species (see Rossatto andKolb 2009). Mature leaves are able to detect and transmitchanges in the external environment to new leaves of the sameplant and induce an appropriate developmental response ofnew leaves (Lake et al. 2001). The production of season-specific leaf types was not previously acknowledged in theplethora of adaptive strategies of the Cerrado plants to dealwith seasonal water availability (Franco 2002). Further studiesare needed to confirm whether the production of season-specificleaf types is of adaptive value for the evergreen species of theCerrado flora.

We found consistent changes in leaf traits along theecologicalgradient, supporting our hypotheses. In accordance to ourpredictions, water economy-related leaf traits increasedtowards canopy openness and light capture-related leaf traitsincrease towards canopy closure. Leaves traits of M. albicansassociated with xeric environments include a well developedcuticle, epidermal cells with straight anticlinal walls on theadaxial surface and sinuous cell walls in the abaxial surface,mesophyll with dorsiventral organisation, sunken stomatarestricted to the abaxial surface and higher trichomeabundance at the abaxial surface. Most of these leaf traits aredocumented in woody species from the Cerrado includingMelastomataceae (Reis et al. 2005). The evolution of thesexeromorphic traits in phylogenetic unrelated species from theCerrado strongly suggests that xeromorphism is thought to have

an adaptive value under conditions of water deficiency, soilacidity and toxicity, among other abiotic selective pressures(Bieras and Sajo 2009).

In this paper we assume that plasticity in water economy traitsand leaf optical properties are directly related to water and lightavailability, respectively (but see Chen et al. 2010). Leavesgrowing under conditions of water deficiency develop smallerbut more densely distributed stomata, thicker wax layers, denserhair cover and smaller SLA (Larcher 1995; Valladares andNiinemets 2008). These adjustments in M. albicans in theharsher environment allow for a more efficient stomatalregulation, increased boundary layer resistance and reduced

transpiration, which in turn, result in more effective gasexchange and higher photosynthetic rate (Dickison 2000;Pearce et al. 2006). Higher palisade parenchyma thickness in

campo sujo probably eases the path of light directed inside themesophyll, allowing for a more uniform photosynthesis rate(Vogelman et al. 1996). On the other hand, lower spongyparenchyma thickness in cerrado leaves is probably due todiffuse light occurrence in this shaded environment (Vogelmanet al. 1996).

Protein-binding capacity rather than well developed cuticlesand a dense trichome cover is negatively correlated with theamountofinsectdamage(Marquis etal. 2002). These polyphenolcomplexes decrease the availability of edible plant tissues andhinder herbivory (Howe and Westley 1990). Despite higherherbivore pressure during the dry season in the Cerrado(Marquis et al. 2001), polyphenols were detected in both dryandrainy seasoninM. albicans leaves. Although calcium oxalatecrystals have been argued to function as a herbivory deterrentthey also have reflective properties that evenly refract lightamong the mesophyll cells, and can also operate in thedetoxification of heavy metals within the cells (Franceschi andNakata 2005).

In conclusion, this study shows high leaf plasticity inM. albicans an obligatory apomictic, widespread shrub. Wehave also found a remarkable seasonal and habitat effect onphenotypic plasticity, confirming that a combination of shadeand drought can pose very strong ecological limits. Theimplications of seasonal-dependent variation in plasticityunder future climate scenarios are currently unknown. Furtherstudies are needed in order to incorporate the consequences of

this type of plasticity limitation into future projections of globalchange.

Acknowledgements

We thank Instituto Chico Mendes at the Estaco Ecolgica de Pirapitinga(Trs Marias, MG) for logistical support and PIBIC/UNA for the scholarship

provided to the first author. We also thank R. M. S. Isaias and L. C. N. Melofor technical help. We thank F. H. A. Vale, G. F. C. Arajo, M. M. Toledo,R. Laia, D. R. Rossatto and F. Valladares for valuable comments on earlyversions of the manuscript. The comments by two anonymous refereessignificantly improved the manuscript quality.

Table 4. Values of relative distance plasticity index of histometric leaf traits of Miconia albicans along a soil

fertility gradient in the Brazilian Cerrado during the dry and rainy season

Histometric trait CS-CE CE-CD CS-CDDry Rainy Dry Rainy Dry Rainy

Cuticle thickness 0.09a 0.17bc 0.05a 0.08a 0.23c 0.28cEpidermis thickness 0.04a 0.09b 0.03a 0.13b 0.02a 0.28d

Palisade thickness 0.16b 0.10b 0.01a 0.30c 0.22bc 0.54dSpongy parenchyma thickness 0.06b 0a 0.01a 0.07b 0.06b 0.14c

Total mesophyll thickness 0.09b 0.08b 0a 0.19c 0.16bc 0.38dAdaxial trichome density 0.08 0.06 0.14 0.25 0 0.15Abaxial trichome density 0.01a 0a 0.01a 0.08b 0.11bc 0.15cStomatal density 0.19a 0.24a 0.02b 0.05b 0.19a 0.21a

CS, campo sujo; CE, cerrado sensu stricto; CD, cerrado. Within rows, means followed by different letters indicatesignificant differences (P< 0.05) between seasons determined by Tukey test.

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Manuscript received 15 October 2010, accepted 17 March 2011

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