High Anatomical and Physiological Leaf Plasticity of Ocotea Odorifera (Lauraceae) in Response to Different Levels of Radiation Availability

Botany

High anatomical and physiological leaf plasticity of Ocotea odorifera (Lauraceae) in response to different levels of radiation availability

Journal: Botany

Manuscript ID cjb-2019-0128.R1

Manuscript Type: Article

Date Submitted by the 14-Jul-2020 Author:

Complete List of Authors: Leme, Gabriele; Universidade Federal de Alfenas, ICN Ramos, Flavio; Universidade Federal de Alfenas, ICN Pereira, Fabricio; Universidade Federal de Alfenas, Instituto de Ciências da NaturezaDraft Polo, Marcelo; Universidade Federal de Alfenas, ICN

Sun leaves, Shade leaves, Shade-tolerant species, Light environments, Keyword: Photosynthetic efficiency

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

1 High anatomical and physiological leaf plasticity of Ocotea odorifera (Lauraceae)

2 in response to different levels of radiation availability

4 Gabriele Marques Leme1, Flavio Nunes Ramos1, Fabricio José Pereira1, Marcelo Polo1*

6 1Laboratório de Ecologia de Fragmentos Florestais (ECOFRAG), Instituto de Ciências

7 da Natureza, Universidade Federal de Alfenas (UNIFAL-MG), Rua Gabriel Monteiro

8 da Silva, n. 700, Alfenas, MG. CEP 37130-001, Brazil. email list:

9 [email protected], [email protected], fabricio.pereira@unifal-

10 mg.edu.br, [email protected] 11 *Author for correspondence: [email protected],Draft phone: +55 35 3701-9681.

12 Abstract

13 Our goal was to investigate leaf morpho-physiological plasticity of Ocotea

14 odorifera trees growing under different environmental conditions in a fragmented

15 forest. Microclimatic data were collected in a pasture matrix, forest edge, and forest

16 interior in three Atlantic Forest fragments. Leaf gas exchange, as well as leaf anatomy

17 in paradermal and transversal sections, were evaluated in individuals in these

18 environments. Radiation intensity and temperature had higher means in the pasture

19 matrix compared to that in both the forest interior and edge. However, internal portions

20 of the canopy did not exhibit significant variation in radiation or temperature. External

21 canopy leaves exhibited higher net photosynthesis in plants from the pasture matrix, but 22 the shaded forest interior favoredDraft this parameter for internal leaves. Variation in net 23 photosynthesis and other gas exchange parameters were related to thinner shade leaves

24 in forest interior individuals and internal leaves with lower stomatal density. Although

25 the pasture matrix, forest edge, and forest interior experienced differences in light and

26 temperature, leaf position in the canopy produced microclimatic variations, which

27 modified gas exchange and anatomy. Thus, O. odorifera shows the potential for

28 reforestation programs because of its high leaf plasticity, which will enable it to

29 overcome light and temperature variation.

31 Keywords: Light environments đ Pasture matrix đ Photosynthesis đ Shade leaves đ

32 Shade-tolerant species đ Sun leaves

33 Introduction

34 Tree communities in pastures in tropical habitats are formed by regeneration or

35 the remaining tree species retained by landowners during forest clearing and subsequent

36 management (Manning et al. 2006; Siqueira et al. 2017). Landowners maintain tree

37 species in pastures for two reasons: (i) shading efficiency (Titto et al. 2011), and (ii)

38 timber quality (Siqueira et al. 2017). However, these trees may die because of

39 environmental changes caused by high irradiation and temperature in pastures as

40 compared to that in forests.

41 The intensity and quality of light, as well as other microclimatic parameters,

42 vary within environments (Wagner et al. 2008, Barros et al. 2012, Reyer et al. 2013) 43 and consequently, may modify theDraft physiology, anatomy, and morphology of leaves 44 (Hogewoning et al. 2010, Macedo et al. 2011). Likewise, plants growing under different

45 light conditions may adjust their photosynthetic apparatus through phenotypic plasticity

46 (Valladares and Niinemets 2008, Wagner et al. 2008), which enables photosynthetic

47 acclimation and higher growth rates (Athanasiou et al. 2010). Moreover, leaves may

48 also exhibit anatomical (Weston et al. 2000) and physiological differences (Murchie and

49 Horton 1997) in response to changes in light intensity. Well-understood differences in

50 anatomy and physiology of sun and shade leaves prevent or mitigate damage (Fan et al.

51 2013).

52 The ability to acclimate to different radiation intensities varies among species

53 (Gratani 2014). Early successional and light-demanding species likely have higher

54 photosynthetic plasticity and acclimation capacity than late-successional and shade-

55 tolerant species (Portes et al. 2010, Longuetaud et al. 2013). However, there is

56 increasing evidence that acclimation potential is not always related to the successional

57 status of the plant species (Wyka et al. 2007, Souza et al. 2008, Kuptz et al. 2010).

58 Krause et al. (2012), for example, found that a late-successional tree species could grow

59 under full exposure to sunlight. Thus, the ability to tolerate high-light environments,

60 such as that in pastures, is species-specific and may represent a selective advantage.

61 Most studies on acclimation processes of plants have been conducted using

62 species exposed to a series of radiation intensity levels (Athanasiou et al. 2010). Few

63 studies have focused on photosynthetic responses of forest species to light conditions in

64 the field (Ramos and Grace 1990, Abrams and Mostoller 1995, Carswell et al. 2000,

65 Domingues et al. 2007), although some studies have investigated the photosynthetic

66 responses of seedlings planted in open pastures and under remnant shrubs and trees 67 (Loik and Holl 1999, 2001). In thisDraft study, we investigated the relationships between leaf 68 anatomy and photosynthetic traits under different light conditions. More precisely, we

69 investigated the plasticity of Ocotea odorifera trees along a radiation gradient.

70 Ocotea odorifera is a vulnerable tree species from the Brazilian Atlantic

71 Forest, which is commonly recommended for shading cattle in pastures. This species

72 develops a canopy that can reach 4œ6 m in diameter and provides substantial shade

73 (Lopes et al. 1996). However, Carvalho (2005) has indicated that O. odorifera requires

74 low to medium shading intensity as a sapling and is classified as a late secondary or

75 shade-tolerant climax species. Additionally, this species does not survive when planted

76 in full exposure to sunlight and there is no report regarding its regeneration in secondary

77 vegetation (Carvalho 2005). However, we determined that adult O. odorifera could

78 survive in pastures and exhibited substantial anatomical and physiological plasticity in

79 its leaves. Our results are important to management and conservation programs where

80 this species is relevant for restoration projects.

81 We hypothesized that photosynthetic responses of O. odorifera were mediated

82 by its leaf anatomy and the microclimate. The results from this study indicated plant

83 tolerance to different radiation conditions and additionally provided insights regarding

84 the suitability of this species for use in restoration programs.

86 Materials and Methods

88 Tree species and study sites

89 Ocotea odorifera (Vell.) Rohwer (Lauraceae), commonly known as sassafras, is

90 a Brazilian native tree species of the Atlantic Rainforest that occurs in the southern and 91 southeastern regions of Brazil andDraft some parts of Bahia State (Carvalho 2005). The 92 species is prized for the quality of its wood but particularly for the essential oil

93 components that contain safrole (Mossi et al. 2013), which is exported to Japan, the

94 United States, and some European countries (Oltramari 2001). Owing to its

95 indiscriminate exploitation, O. odorifera was included in the Brazilian checklist of

96 vulnerable plant species (IBAMA 2008) and was designated as vulnerable according to

97 The World Conservation Union Red List of Threatened Species (Varty 1998). The

98 natural reproduction of O. odorifera presents difficulties because of the time it takes to

99 reach physiological maturity, which is approximately 25 to 40 years, with an average

100 production of 2,000 diaspores/year/plant (Duarte da Silva et al. 2001). Furthermore,

101 fruits are difficult to obtain because of their irregular and biannual development (Inoue

102 et al. 1984). We chose this species because of its ecological importance, its endangered

103 status, and primarily because it appears in three distinct habitats: along forest edges, in

104 forest interiors, and as isolated trees in the pasture matrix near forest fragments.

105 The individuals chosen in our study were no taller than 6.0 m. Data sampling

106 occurred at an average canopy height of 2.0 m from nine individuals, which exhibited

107 good phytosanitary condition. Ocotea odorifera individuals in pastures were remnants

108 from the deforestation process and were left to provide shade for cattle. We considered

109 external leaves as those at the third internode or outer ones, and internal leaves as those

110 at the fourth internode or inner ones. Only fully expanded leaves were collected.

111 The study took place in three forest fragments near Alfenas City (21°25•45ŽS,

112 45°56 •50ŽW, and an altitude of 880 m asl) in southern Minas Gerais, Brazil. This is an

113 Atlantic Forest area with semi-deciduous vegetation and a Cwb type climate, with warm

114 and rainy summers and cold and dry winters. 115 The forest fragments studied wereDraft surrounded by pasture. We investigated three 116 environments with naturally occurring individuals of O. odorifera: (1) the pasture

117 matrix, (2) the forest edge, and (3) the forest interior (at least 200 m from any edge;Fig.

118 1).

119 Microclimatic data

120 To compare the microclimate among the environments, we measured the

121 following variables: (1) air temperature, (2) soil temperature, and (3) irradiance. This

122 experiment was designed as a factorial 2 þ 3 (internal and external leaves þ three

123 environments), and 90 samples were collected during the experiment daily between

124 December 2013 and March 2014 (n = 540). Variables 1 and 2 were measured with a

125 portable digital thermometer and variable 3 was measured with a solar power meter

126 MES-100 (Instrutherm Instrumentos, São Paulo, Brazil). All the measurements were

127 taken at three fixed points in each environment per forest fragment, and the fixed points

128 were represented by O. odorifera trees, with one measurement adjacent to each tree and

129 another below the canopy. These measurements resulted in the following conditions:

130 external pasture leaves, internal pasture leaves, external forest edge leaves, internal

131 forest edge leaves, external forest interior leaves, and internal forest interior leaves. The

132 measurements were limited to morning hours (9:00œ12:00 h), and only on days with

133 similar weather conditions (cloudless sunny days) between December 2013 and March

134 2014.

135

136 Gas exchange measurements

137 Net photosynthesis (Pn), intercellular carbon dioxide concentration (Ci),

138 transpiration rate (E), and stomatal water conductance (gs) were measured with an 139 infrared gas analyzer LI-6400XT Draft (Li-Cor Inc., Lincoln, USA). The temperature was 140 maintained at 28°C in the cuvette (block temperature) and the photosynthetically active

141 photon flux density (PPFD) was fixed in the IRGA chamber at 1400 µmol mœ2 sœ1.

142 Radiation intensity in the cuvette was defined as the average radiation in open

143 environments during the wet season and this light intensity was sufficient to saturate the

144 photosynthetic system and to compare differences between internal and external leaves.

145 All measurements were performed in the field, during morning hours (9:00œ12:00 h)

146 using fully expanded, mature leaves from nodes 2œ5 from the base of plagiotropic

147 branches of the canopy, using leaves lacking evident damage by herbivores or

148 pathogens. Nine individual plants per light environment (forest interior, forest edge, and

149 pasture) were selected at random and gas exchange parameters were measured on 10

150 leaves per plant, of which five were shade leaves (inner canopy) and the other five were

151 sun leaves (outer canopy). We considered external leaves as those at the third internode

152 or outer ones and internal leaves as those at the fourth internode or inner ones. Only

153 fully expanded leaves were collected. These measurements resulted in procurement of

154 leaves of the following types: external pasture leaves, internal pasture leaves, external

155 forest edge leaves, internal forest edge leaves, external forest interior leaves, and

156 internal forest interior leaves.

157

158 Leaf quantitative anatomy

159 For anatomical analysis, we collected three fully expanded leaves per position in

160 the canopy (external and internal) at the 3rd to 4th nodes at the base of plagiotropic

161 branches of nine trees. Leaves for anatomical analysis were sampled in two separate

162 experiments. The first experiment compared external and internal leaves from plants 163 grown in the pasture matrix only, andDraft the second experiment compared leaves (external 164 ones only) from the three environments (pasture matrix, forest edge, and forest interior).

165 During the experiment, microclimatic data showed no significant change in radiation

166 intensity for internal and external areas for the forest edge and interior, whereas the

167 pasture matrix clearly showed higher means for the external leaves. Thus, comparison

168 of external and internal leaf anatomy was possible for pasture matrix plants only

169 because the forest edge and interior leaves were unlikely to develop into sun and shade

170 leaves depending on the leaf position in the canopy. Leaf samples were fixed in FAA

171 for 72 h and then stored in 70% ethanol. Samples were dehydrated by a graded ethanol

172 series and embedded in resin according to the manufacturer's instructions (Leica

173 Microsystems, Wetzlar, Germany). Sections (12 µm thick) were cut using an HM 340E

174 rotary microtome (Thermo Fisher Scientific, Shanghai, China). The sections were

175 affixed to glass slides and stained for 5 min in 0.06% toluidine blue solution. We

176 examined three sections per slide with a light microscope AxioLab.A1 (Carl Zeiss

177 Microscopy GmbH, München, Germany) and photographs were taken using an attached

178 digital camera Axiocam ERc 5s (Carl Zeiss Microscopy GmbH, München, Germany).

179 From the interveinal region in the cross-sections of three randomly selected microscopic

180 fields, we measured the following anatomical traits: abaxial and adaxial epidermal

181 thickness (ETab and ETad, respectively), palisade parenchyma thickness (PT), spongy

182 parenchyma thickness (ST), mesophyll thickness (Tm), and total leaf blade thickness

183 (LT), totaling 54 data entries per environment (pasture, forest interior, and forest edge)

184 or canopy position (external and internal). Leaf area from internal and external leaves

185 was obtained with a leaf area scanner (CI-20, CID Bio-Science, WA USA) and 40

186 leaves were sampled for each leaf type (n = 80). 187 The leaves of O. odoriferaDraft are hypostomatous. Thus, we calculated stomatal 188 density (SD) by taking impressions of the abaxial surface of the leaf epidermis at the

189 widest point of the leaf near the midrib by printing with an instant adhesive

190 (methacrylate ester). For the impressions, one leaf was selected from each tree, and the

191 same trees used for other measurements totaling nine leaves per environment (forest

192 interior, forest edge, and pasture). All impressions were fixed on glass slides and

193 examined under a light microscope and imaged as described above. Three microscopic

194 fields from each slide were randomly selected. Stomata were counted and SD was

195 calculated as the number of stomata per unit of leaf area (expressed as stomata mm-×).

196

197 Statistical analysis

198 Microclimate and gas exchange data were analyzed using a two-way ANOVA

199 and both datasets were further compared using the Scott-Knott‘s test at p <0.05. We

200 compared the leaf anatomy data using a one-way ANOVA followed by the Tukey‘s

201 multiple comparisons at p<0.05 because data for internal and external leaves were only

202 collected in the pasture matrix. Thus, there were two experiments for anatomical data:

203 one compared the effect of internal and external leaves from the pasture matrix plants (I

204 þ E), and the second compared the effects of three environments (pasture matrix þ

205 forest edge þ forest interior). All data exhibited a normal distribution and

206 homoscedasticity as assessed by the D‘Agostino-Pearson tests and Levene tests,

207 respectively.

208

209 Results

210 Summarized data from ANOVA analyses are shown in Table 1. For 211 microclimate and gas exchange parameters,Draft a significant interaction occurred between 212 factors (leaf positions and environments). Data for leaf anatomy are shown separately

213 for the two experiments.

214 Microclimatic data

215 Radiation intensity was higher in areas near external leaves in plants from the

216 pasture matrix and forest edge. However, no significant difference in radiation intensity

217 occurred for plants in the forest interior environment (Table 2). Additionally, radiation

218 near external leaves had higher means in the pasture matrix and the lowest occurred in

219 the forest interior. However, in the forest interior, levels did not vary between external

220 and internal leaves (Table 2).

221 Higher air and soil temperatures occurred near external leaves in the pasture

222 matrix and forest edge environments but no significant differences occurred in the forest

223 interior (Table 2). Additionally, soil temperatures were higher in the pasture matrix and

224 forest edge as compared to that in the forest interior for both external and internal leaf

225 locations (Table 2). However, the air temperature was highest in the pasture matrix in

226 external leaf locations followed by the forest edge. The lowest means occurred for the

227 forest interior; however, measurements taken near internal leaves had lower means for

228 the pasture matrix compared to that of the forest edge and forest interior (Table 2).

229 Gas exchange parameters

230 Net photosynthesis was 32.4% higher in plants in the pasture matrix compared to

231 that of forest edge and interior for external leaves (Table 3). Interestingly, the internal

232 leaves exhibited higher net photosynthesis (34.4%) in plants from the forest interior

233 compared to that of the pasture matrix and forest edge (Table 3). Additionally, external

234 leaves had net photosynthesis that was 54.2% higher compared to that of internal leaves 235 on plants from the pasture matrixDraft and forest edge. However, plants from the forest 236 interior exhibited no significant differences in net photosynthesis between internal and

237 external leaves (Table 3).

238 Stomatal conductance varied for O. odorifera leaves among the environments

239 (Table 3). Both external and internal leaves showed lower stomatal conductance in

240 shaded environments. The highest stomatal conductance occurred for plants from the

241 pasture matrix and the lowest means occurred for plants from the forest interior. Forest

242 edge plants showed intermediate mean values for stomatal conductance. Additionally,

243 stomatal conductance was higher (50%) on external leaves in the pasture matrix and

244 forest edge, although no differences occurred for plants from the forest interior (Table

245 3).

246 The transpiration rate for both external and internal leaves was highest for plants

247 from the pasture matrix; plants from the forest edge showed intermediate values and the

248 lowest values were recorded for forest interior plants (Table 3). Additionally, external

249 leaves exhibited higher transpiration in all environments compared to that of the internal

250 leaves (Table 3).

251

252 Anatomical leaf measurements

253 External and internal leaves distributed in the three environments exhibited

254 differences for most leaf anatomical characteristics. The external leaves had a thicker

255 epidermis, as well as PT and ST, Tm, and whole leaf thickness compared to the values

256 for internal leaves, whereas internal leaves showed higher SD and leaf area (Table 4).

257 Regarding plants in the three environments, plants from the pasture matrix exhibited the

258 highest values for all anatomical variables (Table 4). However, plants from forest 259 interior and forest edge displayed noDraft significant differences for leaf thickness, as well as 260 both adaxial and abaxial epidermis and ST (Table 5). Furthermore, both the Tm and PT,

261 as well as the SD, were lowest for the plants from the forest interior compared to those

262 in other environments (Table 5).

263

264 Discussion

265 Differences in light intensity and temperature between environments were

266 expected. In tropical forests, the shading effect of the canopy may reduce the photon

267 flux density within a forest fragment (Rossato et al. 2010, Venturoli et al. 2012). The

268 leaf area of a canopy is strongly and negatively correlated with light intensity inside a

269 forest fragment (Keeling and Phillips 2007). Therefore, our data corroborate the shading

270 effect inside forest fragments and this is likely related to lower temperatures in the

271 forest interior. However, the novel results of our study demonstrated that higher

272 radiation intensity occurred only for the external leaves of the canopy because internal

273 leaves showed no modification in radiation intensity caused by self-shading promoted

274 by the canopy. Thus, plant distribution along the radiation gradient in forest fragments

275 and the pasture matrix depends on the capacity of leaves to adapt to different parts of

276 the canopy and maintains photosynthetic activity at optimal levels. All the microclimate

277 changes lead to responses in the physiology and anatomy of plants and these

278 modifications can be used as markers to understand plant distribution among

279 environments.

280 The increased Pn under higher light intensity has been well-documented in

281 previous studies for various plant species (Portes et al. 2010, Lobos et al. 2012,

282 Longuetaud et al. 2013). Higher light availability may enhance net photosynthesis 283 (Zhou and Han 2005) and this increaseDraft is related to leaf anatomical modifications. 284 Thicker leaves are produced under higher radiation intensity (Fan et al. 2013, Scoffoni

285 et al. 2015) and a direct correlation occurs between thicker leaves and higher

286 chlorophyll content, photosynthetic tissues, and enzymes (Oguchi et al. 2003,

287 Terashima et al. 2011).

288 The lower light intensity inside the forest fragments reduced the photosynthesis

289 of O. odorifera. Therefore, O. odorifera from the forest interior had lower Pn values

290 because their leaves were thinner and the light intensity reaching this environment was

291 strongly reduced compared to that in the pasture matrix and forest edge environments.

292 However, this species shows high leaf anatomical and physiological plasticities, which

293 are related to different internal leaf structures compared to those in external leaves.

294 Leaves of O. odorifera were different within the canopy. A higher

295 photosynthetic potential of sun leaves compared to shade leaves has been reported for

296 different plant species (Rossato et al. 2010, Vieira et al. 2015). The external leaves of O.

297 odorifera could be compared to sun leaves and the internal leaves were equivalent to

298 shade leaves; however, this distinction depended on the environment where the plants

299 grew because individuals from forest interior produced shade leaves only.

300 The enhanced chlorenchyma thickness of external leaves was correlated with the

301 number of chloroplasts, Rubisco, and chlorophyll content, which have been reported to

302 improve light usage and CO2 fixation (Ivancich et al. 2014, Zivcak et al. 2014). In

303 addition to thicker photosynthetic tissues, external leaves of O. odorifera also exhibited

304 higher stomatal conductance. Higher photosynthesis promoted by increased stomatal

305 conductance and CO2 uptake in leaves is supported by the findings of previous studies

306 (Sack et al. 2006, Santiago and Kim 2009, Lawson et al. 2011). As they were exposed 307 to high radiation levels in the pastureDraft matrix, the external leaves had to prevent 308 overheating, which might be achieved through an increase in transpiration rate.

309 Therefore, higher radiation intensity for external leaves reduced resistance to CO2

310 uptake and increased the thickness of photosynthetic tissues, thereby leading to higher

311 photosynthetic potential.

312 In O. odorifera internal leaves, their lower photosynthesis may be partially

313 related to photoinhibition at higher light intensities. The excessive light intensity may

314 lead to limitations in the photosynthetic apparatus by photoinhibition (Yamamoto et al.

315 2014) and this inhibition is related to chloroplast and thylakoid membrane structure

316 (Tsabari et al. 2015). The internal leaves were exposed to the same saturating radiation

317 level as the sun leaves (1400 µmol m-2s-1), but these leaves exhibited photoinhibition

318 because of their thinner photosynthetic tissues. Regarding the Pn limitation in O.

319 odorifera growing in the forest interior, another factor in this environment may limit

320 CO 2 uptake. CO2 availability is one of the most important components of

321 photosynthesis efficiency (Zhou and Han 2005). Additionally, SD is positively

322 correlated with stomatal conductance (Baroli et al. 2008); thus, higher SD produces

323 higher stomatal conductance (Lee et al. 2007). The lower stomatal conductance and

324 density in O. odorifera leaves from forest interior individuals may limit photosynthesis.

325 However, internal leaves showed higher net photosynthesis in the forest interior as

326 compared to that of other environments. This may also be part of the anatomical

327 plasticity observed in these plants because thinner leaves are adapted to diffuse radiation

328 in shade environments (Li et al. 2014). Therefore, the O. odorifera crown exhibits

329 different leaf types: shade leaves internally and sun leaves externally. This is an

330 important trait for cosmopolitan trees and permits this species to adapt to both the 331 original forest interior sites, as wellDraft as open areas formed by forest fragmentation. This 332 aids in understanding the wide geographical distribution of O. odorifera (Oltramari,

333 2001) because anatomical plasticity is one of the key features for plants that can

334 overcome changes in light availability and maintain photosynthetic activity (Valladares

335 and Niinemets 2008, Wagner et al. 2008). Additionally, both anatomical and

336 photosynthetic plasticities are relevant for species used in reforestation programs and

337 may help to restore different environments. This is important because the reforestation

338 process changes light intensity and promotes a shading effect that gradually increases

339 across time as the environment is restored. Species such as O. odorifera, which exhibit a

340 capacity to develop shade and sun leaves, are important in overcoming these light

341 intensity changes during the reforestation process and it is necessary to pay more

342 attention to the anatomical features of these species. Furthermore, O. odorifera is an

343 endangered species, and information regarding its photosynthetic responses and

344 plasticity in different environments can contribute to its conservation.

345 Light intensity in open environments can reach values of up to 1400 µmol m-2 s-1

346 or higher; however, forest fragment reduces radiation intensity to 10% of that measured

347 in open environments (Venturoli et al. 2012). Therefore, the radiation used in the IRGA

348 chamber was intentionally fixed at 1400 µmol m-2 s-1. Since this species is found in all

349 environments tested, its anatomical and physiological plasticity promote species

350 adaptation. Although plant species may show plasticity to light intensities (Portes et al.

351 2010, Longuetaud et al. 2013, Gratani 2014), a light intensity of 1400 µmol m-2s-1 (close

352 to the pasture matrix condition) may lead to photoinhibition of forest interior leaves. In

353 general, O. odorifera forest interior leaves showed high plasticity and Pn values that

354 were only slightly lower than those for leaves from the pasture trees. 355 Our results showed that O. Draftodorifera trees growing in contrasting environmental 356 conditions, such as pastures, forest fragment edges, and forest interiors, could survive

357 because of their anatomical and physiological plasticity. Polylepis cuadrijuga, an

358 endemic tree species in Colombia, also exhibits plasticity in anatomical and

359 physiological behavior in pastures and forest edges in fragmented environments (Ramos

360 et al. 2013). Montgomery (2004) studied three shade-tolerant tree species growing

361 across a light gradient created by a forestœpasture edge in Costa Rica and demonstrated

362 that species differed in the magnitude and plasticity in growth. One tree species had low

363 relative growth rates (RGR) and low plasticity, but the other two species presented

364 higher RGR and plasticity (Montgomery 2004). Our study indicates that plasticity in

365 photosynthetic physiology is very important to the variation in tree growth and survival

366 in habitats with contrasting light availability. Thus, O. odorifera leaves demonstrated

367 wide plasticity between truly shaded leaves and sun leaves developing on trees

368 subjected to different light intensities. This explains, to some extent, the wide

369 distribution of this species among different habitats in natural areas.

370

371 Conclusion

372 Our study indicates that O. odorifera has high anatomical and physiological

373 plasticity and has the potential to acclimate and grow under different light conditions

374 and survive in both shade and open environments. The pasture matrix, forest edge, and

375 forest interior environments had different light and temperature conditions. However,

376 these conditions varied with leaf position in the canopy. The net photosynthesis of O.

377 odorifera was higher on external leaves in richer light environments (pasture matrix), 378 but could be higher in internal leavesDraft in environments with lower light intensities (forest 379 interior) . Environments with higher light intensities promoted the development of

380 thicker external leaves with higher net photosynthesis; however, the thinner internal

381 leaves were favored in the shaded forest interior and exhibited higher net photosynthesis

382 in this environment. Therefore, this late secondary to climax forest species successfully

383 copes with the changing environment, thus showing potential for forest restoration

384 systems.

385

386

387 Acknowledgements

388 We are grateful to Eduardo L. Abreu, Vinícius de Toledo and Gabriel A. Diniz for the

389 valuable help with the fieldwork, to Peter E. Gibbs for the English revision, and to

390 Erica Hasui for the map confection. The authors are also grateful to the Universidade

391 Federal de Alfenas-MG, Fundação de Amparo à Pesquisa do Estado de Minas Gerais

392 (FAPEMIG), and Companhia Vale do Rio Doce (VALE) (CRA-RDP-0104-10) G.M.L.

393 This study was financed, in part, by the Coordenação de Aperfeiçoamento de Pessoal de

394 Nível Superior - Brasil (CAPES) - Finance Code 001, and CNPq, which provided

395 scholarship and research support.

396

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650 Table 1. Summarized ANOVA results for all variables analyzed, including mean square 651 values, F test results, and P values. CV% = coefficient of variation.

Variable CV% Mean F test P value square value values Radiation intensity 34.21 8072974.60 732.864 <0.0001 Air temperature 4.52 104.35 71.91 <0.0001 Soil temperature 3.42 23.24 39.49 <0.0001 Net photosynthesis 18.19 42.80 9.75 <0.0001 Stomatal conductance 1.35 0.008 10.61 <0.0001 Transpiratory rate 15.17 2.40 5.52 0.0041 Abaxial epidermis thickness (IxE) 16.04 31.50 18.14 <0.0001 Abaxial epidermis thickness 14.37 54.90 46.07 <0.0001 (PMxFExFI) Adaxial epidermis thickness (IxE) Draft17.93 131.53 38.30 <0.0001 Adaxial epidermis thickness 14.15 256.27 160.69 <0.0001 (PMxFExFI) Leaf area (IxE) 27.16 3274.15 17.93 <0.0001 Leaf area (PMxFExFI) 27.62 3177.69 20.12 <0.0001 Leaf thickness (IxE) 8.89 22597.77 20.32 <0.0001 Leaf thickness (PMxFExFI) 11.29 26440.70 50.44 <0.0001 Mesophyll thickness (IxE) 9.44 8030.91 22.062 <0.0001 Mesophyll thickness (PMxFExFI) 10.51 23938.32 61.79 <0.0001 Palisade parenchyma thickness (IxE) 17.23 12040.51 129.49 <0.0001 Palisade parenchyma thickness 16.13 10892.36 161.72 <0.0001 (PMxFExFI) Spongy parenchyma thickness (IxE) 12.99 2238.96 6.27 0.0138 Spongy parenchyma thickness 16.31 1288.16 2.64 0.0431 (PMxFExFI) Stomatal density (IxE) 20.66 135981.12 29.59 <0.0001 Stomatal density (PMxFExFI) 21.24 132725.79 31.49 <0.0001

652 P -value limit of the software is 0.0001, results lower than this limit are indicated as 653 p<0.001. For anatomical data = (IxE) = internal þ external leaf comparison; 654 (PMxFExFI) = pasture matrix þ forest edge þ forest interior.

Draft

662 Table 3. Gas exchange of Ocotea odorifera leaves from the external and internal parts

663 of the canopy from plants growing in forest fragments with different environments

664 provided by surroundings. Data are shown as the mean ± standard deviation ( n = 54).

-2 -1 Net photosynthesis (µmolCO2 m s ) Environments Leaf types

External leaves Internal leaves

Pasture matrix 3.80±0.9 aA 2.43±1.2 bB Forest edge 3.17±1.4 bA 2.09±0.6 bB Forest interior 2.86±0.4 bA 2.81±0.3 aA -2 -1 Stomatal conductance (molCO2 m s ) Environments Leaf types ExternalDraft leaves Internal leaves Pasture matrix 0.08±0.002 aA 0.06±0.002 aB Forest edge 0.07±0.003 bA 0.04±0.001 bB Forest interior 0.03±0.001 cA 0.02±0.001 cA -2 -1 Transpiratory rate (mmolH2O m s ) Environments Leaf types

External leaves Internal leaves

Pasture matrix 1.89±0.4 aA 1.45±0.5 aB Forest edge 1.63±0.7 bA 1.14±0.2 bB Forest interior 0.80±0.3 cA 0.63±0.2 cB

665 Different uppercase letters in the same row and lowercase letters in the same column

666 indicate significant differences at p<0.05 (Scott-Knott‘s test).

Table 4 Anatomy of leaves from Ocotea odorifera at different positions in the canopy

(external and internal leaves). Data are shown as the mean ± standard deviation (n =

27).

Variables External leaves Internal leaves

ADE (µm) 11.44 ± 0.24a 9.23 ± 0.26b

ABE (µm) 8.76 ± 0.21a 7.68 ± 0.15b

PP (µm) 66.51 ± 1.74a 45.39 ± 0.65b

SP (µm) 140.98 ± 2.54b 150.09 ± 2.60a

MT (µm) 210.78 ± 2.01a 193.53 ± 3.07b

LT (µm) 227.68 ± 2.52a 212.39 ± 2.96b

SD (mm×) 371.58Draft ± 12.70a 284.67 ± 9.70b

LA (mm×) 41.16 ±1.37b 51.87 ±1.50a

ADE, adaxial epidermis thickness; ABE, abaxial epidermis thickness; PP, palisade parenchyma thickness; SP, spongy parenchyma thickness; MT, mesophyll thickness;

LT, leaf thickness; SD, stomatal density; LA, leaf area. Different letters in the same row indicate significant differences at p<0.05 (Tukey‘s test).

Table 5. Anatomy of leaves from Ocotea odorifera growing in three habitats differing

in light interception: forest interior, forest edge, and pasture matrix. Data are shown as

the mean ± standard deviation (n = 27).

Variables Pasture matrix Forest Edge Forest Interior

ADE (µm) 10.13 ± 0.30a 7.72 ± 0.11b 7.62 ± 0.13b

ABE (µm) 8.72 ± 0.20a 6.92 ± 0.09b 7.12 ± 0.13b

PP (µm) 58.86 ± 2.02a 47.32 ± 0.66b 38.78 ± 0.54c

SP (µm) 150.86 ± 2.41a 133.57 ± 3.19b 131.76 ± 3.24b

MT (µm) 207.72 ± 2.71a 181.08 ± 2.67b 170.07 ± 3.22c

LT (µm) 228.64 ± 3.06a 195.53 ± 3.13b 185.27 ± 3.60b

SD (mm×) 361.48 ± 12.26Drafta 304.56 ± 13.89b 222.04 ± 2.85c

ADE, adaxial epidermis thickness; ABE, abaxial epidermis thickness; PP, palisade

parenchyma thickness; SP, spongy parenchyma thickness; MT, mesophyll thickness;

LT, leaf thickness; SD, stomatal density. Different letters in the same row indicate

significant differences at p<0.05 (Tukey‘s test).

667

Draft

668 669 Figure 1. Map indicating three forest fragments nearby Alfenas city, southern Minas

670 Gerais, Brazil where have naturally occurring individuals of O. odorifera. This map was

671 made with ArcGIS 10.5 software, based on the Rezende et al. (2018) map and the

673 Google).

674