Traços Funcionais Foliares na Amazônia

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1 FUNCTIONAL LEAF TRAITS IN CONGENERIC SPECIES OF TREE 
2 COMMUNITIES IN AN EDAPHIC GRADIENT IN CENTRAL AMAZON
3
4 Lucas Vinicius Cavalcante Esteves1*, Kleyton Kleber dos Santos Correa1, Mayda Cecília 
5 dos Santos Rocha6, Gleicy Assunção Rodrigues3, Darlisson Mesquita Batista1, Anderson 
6 Alves-Araújo4, Marina Magalhães Hirota5,7, Deliane Vieira Penha1, Leandro Lacerda 
7 Giacomin2, Advanio Inácio Siqueira-Silva1,6 
8 * Corresponding author
9 1 – Graduate Program in Biodiversity, Federal University of Western Pará, Santarém (PA), Brazil;
10 2 – Federal University of Paraíba, João Pessoa (PB), Brazil;
11 3 – Graduate Program in Botany, National Institute of Amazonian Research, Manaus (AM), Brazil;
12 4 – Institute of Biology, Federal University of Bahia, Bahia (BA), Brazil;
13 5 – Federal University of Santa Catarina, Florianópolis (SC), Brazil;
14 6 – Institute of Water Sciences and Technology, Federal University of Western Pará, Santarém (PA), 
15 Brazil;
16 7 – Serrapilheira Institute, Brazil.
17
18 HIGHLIGHTS
19  Variations in the soil provide morphophysiological adjustments in the leaves.
20  Forest species exhibited acquisitive strategies in resource utilization.
21  Savanna species present conservative strategies in resource utilization.
22  Edaphic conditions in the phytophysiognomies modulate leaf functional traits.
23
24 ABSTRACT: Environmental changes are altering global climate and rates of plant 
25 biodiversity. It is predicted that, as a result of these changes, forest formations may 
26 become more open in the future. The objective of this study was to investigate possible 
27 variations in functional leaf traits of congenetic species of Tachigali and Pouteria in tree 
28 communities under different edaphic conditions in savanna and forest areas in the Central 
29 Amazon. Healthy, fully expanded leaves of woody congenetic species were collected in 
30 the phytophysiognomies of Ombrophilous Forest and Amazonian Savanna, both located 
31 in the state of Pará, Brazil. In the field, leaves were collected for subsequent evaluation 
32 of hydraulic traits. For the analysis of anatomical (structural characterization, 
33 micromorphometry, and histochemistry) and morphological traits, the median third of the 
34 leaf blade was selected, and the samples were fixed in FAA70 solution and stored in 70% 
35 ethanol. Leaf samples for anatomical analysis were dehydrated in an increasing ethanol 
36 series, pre-infiltrated, infiltrated, and embedded in resin, transversely sectioned using a 
37 manual rotary microtome (5 μm), stained with toluidine blue, and mounted in acrylic 
38 varnish. Leaf structure observations and records were made using a photomicroscope. 
39 The results showed that congenetic species of Tachigali and Pouteria exhibited 
40 significant variations in functional morphoanatomical leaf traits in the different 
41 phytophysiognomies studied, which likely aid in their growth and establishment. It was 
42 also observed that species present in the savanna environment, with lower nutrient 
43 availability, exhibited more conservative characteristics related to water storage and 
44 drought resistance. However, in species from the forest area, with higher nutrient 
45 availability, more acquisitive strategies were observed. The study suggests that variations 
46 in leaf traits are essential for the survival and development of tree species in each type of 
47 vegetation, and that there is a clear degree of xeromorphism in savanna areas. Therefore, 
48 in the two phytophysiognomies studied, the functional traits of Tachigali and Pouteria 
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49 may be responses to low soil nutrient content, water scarcity, and/or high solar radiation. 
50 Further studies are needed for a better understanding of these adjustments.
51 Keywords: Leaf anatomy; Amazon domain; Ombrophilous Forest; soil gradient; plant-
52 soil relationships; Amazonian Savanna.
53 1. INTRODUCTION
54 Understanding how climate and environmental changes influence plant 
55 biodiversity rates and species distribution is essential in ecology (Condit et al., 2000; 
56 Russo et al., 2005; Baltzer et al., 2009). Environmental factors such as soil 
57 physicochemical properties can act as filters that modulate the floristic and functional 
58 composition of tree communities (Araújo et al., 2022; Elias et al., 2019; Liu et al., 2016; 
59 Pinheiro et al., 2018; Viani et al., 2014). Elucidating how abiotic factors, particularly soil 
60 physicochemical properties, influence the regulation of forest and savanna distribution is 
61 crucial because understanding leaf morphological, anatomical, chemical, and 
62 physiological characteristics is essential for comprehending plant adaptation mechanisms 
63 to future environmental conditions. In this context, the ability of plants to adjust 
64 functional attributes, which are any characteristics directly influencing their performance, 
65 can determine their success in the face of environmental changes since these attributes 
66 influence the capacity of tree species to acquire, conserve, and utilize resources 
67 (Hoffmann et al., 2005; Violle et al., 2007; Wright et al., 2004).
68 Vegetation in savanna and forest areas is strongly influenced by edaphic factors, 
69 where soil nutrient availability affects the functional aspects of plant species, such as 
70 reproduction and growth rates (Lambers et al., 2008; Neves et al., 2022; Pinheiro et al., 
71 2018; Somavilla et al., 2014). Analyzing functional attributes in tropical tree communities 
72 is an important tool that can predict and aid in understanding the composition and 
73 functioning of local or regional communities (Cadotte et al., 2009; Lin et al., 2021; Viani 
74 et al., 2014). However, most investigations on how environmental filters affect the 
75 floristic and functional composition of tree communities have been conducted separately 
76 in savanna and forest areas (e.g., Bona et al., 2020; Neves et al., 2022; Rossatto et al., 
77 2015). Few studies have evaluated the influence of the edaphic gradient on tree species 
78 through a functional approach (Araújo et al., 2021), despite its importance not only in 
79 connecting plant characteristics with environmental factors but also in understanding 
80 plant properties and community structure.
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81 The Phytogeographic Domain of the Amazon is heterogeneous, with open and 
82 forest vegetation formations, and harbors a wide diversity of species, being considered 
83 the largest tropical forest in the world (Cardoso et al., 2017; Ter Steege et al., 2020), 
84 characterized by high functional diversity (Asner et al., 2014). This wide diversity is the 
85 result of a combination of genetic variation and phenotypic plasticity resulting from 
86 environmental conditions over space and time (Bradshaw, 1965, 2006). The Amazon also 
87 presents different phytophysiognomies, defined by climatic, altitudinal, and edaphic 
88 variations (Rossato et al., 2016). In the Amazon Savanna phytophysiognomy, woody 
89 species occur in poor soils and therefore tend to have leaves with lower nutrient 
90 concentration, being considered more conservative plants as they develop more durable 
91 and/or more resistant leaves to herbivores or other injuries (Rossatto et al., 2015; 
92 Somavilla et al., 2014; Wright et al., 2004b). The leaf structure of tree species in the 
93 Amazon Savanna is generally characterized by small, thick, and compact leaves, 
94 amphistomatous, with a thick cuticle, heterogeneous dorsiventral mesophyll, and high 
95 trichome density, evidencing a drought avoidance strategy (Araújo et al., 2021a; Galméscrystals in P. procera, P. reticulata, P. baehniana, 
824 P. bangii, P. ramiflora, and P. guianensis, respectively. Legend: (Ead) adaxial epidermis, (Eab) abaxial 
825 epidermis, (Hip) hypodermis, (Pp) palisade parenchyma, (Pl) spongy parenchyma, (Fv) vascular bundle, 
826 (FF) bundle fibers, (crystal inclusions) double arrowheads, (tannin idioblast) single arrowhead, (cuticle) 
827 arrow. Scale bars: (A-C, D, F, H, and J-L) = 20 µm and (E, I) = 10 µm.
828 Fig. 5. Leaf hydraulic attributes of congenetic woody species from forest and savanna along an edaphic 
829 gradient in Central Amazonia. A: Stomatal density. B: Stomatal index. A total of 10 pairs of congenetic 
830 species associated with contrasting habitats were evaluated ('Forest' areas with soil showing higher nutrient 
831 concentration, with species from this environment considered more resource-acquisitive plants, and 
832 'Savanna' areas with soil showing lower nutrient concentration, with species from this environment 
833 considered more resource-conservative plants). The lines connect pairs of congenetic species, and the 
834 significance values for the paired t-test are shown for each comparison. All relationships are shown in the 
835 original units of measurement. The colors indicate our ten congenetic pairs (yellow, P. procera e P. 
836 ramiflora; green, P. procera e guianensis; orange, P. reticulata e P. ramiflora; blue, P. reticulata e P. 
837 guianensis; pink, P. bangii e P. ramiflora; navy blue, P. bangii e P. guianensis; red, P. baehniana e P. 
838 ramiflora; olive green; black, P. guianensis e P. baehniana; T. chrysophylla e T. vulgaris; purple T. alba e 
839 T. vulgaris). 
840 Fig. 6. Functional attributes of leaf morphology (internal and external) of congenetic woody species from 
841 forest and savanna along an edaphic gradient in the Central Amazon. (A) Leaf thickness. (B) Cuticle 
842 thickness on the adaxial surface of the leaf. (C and D) Epidermal thickness on the adaxial (Ead) and abaxial 
843 (Eab) surfaces of the leaf, respectively. (E and F) Spongy and palisade parenchyma thickness, respectively. 
844 For the 10 pairs of congenetic species associated with contrasting habitats ('Forest' represents areas with 
845 higher nutrient concentration in the soil, with species from this environment considered more resource-
846 acquisitive plants, and 'Savanna' represents areas with lower nutrient concentration in the soil, with species 
847 from this environment considered more resource-conservative plants). The lines connect congenetic species 
848 pairs, and the significance values for the paired t-test are shown for each comparison. All relationships are 
849 shown in original measurement units. The colors indicate our 10 congenetic pairs (yellow, P. procera e P. 
850 ramiflora; green, P. procera e guianensis; orange, P. reticulata e P. ramiflora; blue, P. reticulata e P. 
851 guianensis; pink, P. bangii e P. ramiflora; navy blue, P. bangii e P. guianensis; red, P. baehniana e P. 
852 ramiflora; olive green; black, P. guianensis e P. baehniana; T. chrysophylla e T. vulgaris; purple T. alba e 
853 T. vulgaris). 
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854 Fig. 7. Principal Component Analysis (PCA) of the functional leaf traits of congenetic species of Tachigali 
855 (n=3 for T. chrysophylla, T. alba, and T. vulgaris) and Pouteria (n=3 for P. ramiflora, P. reticulata, P. 
856 procera, P. bangii, and P. guianensis) in the Savanna and Rainforest phytophysiognomies along an edaphic 
857 gradient.
858 Fig. 8. Principal Component Analysis (PCA) of the physicochemical composition of soil in the 
859 phytophysiognomies of Savanna and Ombrophilous Forest along an edaphic gradient in the Central 
860 Amazon. (pH) hydrogen potential; (Ca) Calcium; (P) Phosphorus; (Org. matter) Organic matter; (N) 
861 Nitrogen; (Al) Aluminum; (Mg) Magnesium; (P rem) Removed Phosphorus; (K) Potassium; (Na) Sodium; 
862 Clay, Silt, Coarse sand, Fine sand. 
863 Fig. A. Climatic data from the study area. 1- Ombrophilous Forest in the municipality of Belterra, Pará, 
864 Brazil. 2- Amazonian Savanna area in the municipality of Santarém, Pará, Brazil. 
865 Fig. B. Frontal view of species belonging to the genus Tachigali along an edaphic gradient in the Central 
866 Amazon. (1, 8) Tachigali chrysophylla, (2, 5, 6) T. alba and (3, 7, 8) T. vulgaris. A-C: Adaxial leaf surface. 
867 4-8: Abaxial leaf surface. F, H. Detail of stomata of T. alba and T. vulgaris, respectively. Legend: (Tt) 
868 Tector trichome, (SC) Scar of the base of tector trichomes. Scale bars: (1-5, G) = 20 µm e (6, 8) = 10 µm.
869 Fig. C. Frontal view of the leaf surface of species belonging to the genus Pouteria along an edaphic gradient 
870 in the Central Amazon. (1-17) Pouteria procera, (2, 6, 7) P. reticulata, (9, 12, 13) P. baehniana, (10, 14, 
871 15) P. bangii, (3, 8) P. ramiflora e (K, P, Q) P. guianensis. 1-3, 9-11: Adaxial surface of the leaf. 4-8, 12-
872 16: Abaxial surface of the leaf. 5, 7, 13, 15, 16; 1-3, 4, 7, 8 e 10-12. Stomatal detail of P. procera, P. 
873 reticulata, P. baehniana, P. bangii e P. guianensis, respectively. Legend: (Tt) Tector trichome, (SC) Scar 
874 of the base of tector trichomes. Scale bars: (1, 2, 3, 4, 6, 8, 9, 10, 11, 12, 14, 15, 16) = 20 µm and (5, 7, 13, 
875 15, 16) = 10 µm.
876 Fig. D. Histochemistry for phenolic compounds, lipids, and carbohydrates in leaves of congeneric species 
877 of Tachigali (cross-section) along an edaphic gradient in the Central Amazon. (1-9) T. chrysophylla, (2-5-
878 8) T. alba, (3, 6, 9) T. vulgaris. 1-3: est for phenolic compounds. 4-6: Test for lipids. 7-9: Test for starch. 
879 Legend: (Phenolic compound) asterisk, (Cuticle) arrow, (Starch) arrowhead. Scale bars: (1-9) =20 µm.
880 Fig. E- Histochemistry for phenolic compounds in leaves of congeneric species of Pouteria (transverse 
881 section) along an edaphic gradient in the Central Amazon. (1) Pouteria procera, (2) P. reticulata, 3) P. 
882 ramiflora, (4) P. baehniana, (5) P. bangii, (6) P. guianensis. Legend: (Phenolic compound) asterisk. Scale 
883 bars: (1-9) = 20 µm.
884 Fig. G. Histochemistry for starch in leaves of congeneric species of Pouteria (cross-section) along an 
885 edaphic gradient in the Eastern Amazon. (1) Pouteria procera, (2) P. reticulata, (3) P. ramiflora, (4) P. 
886 baehniana, (5) P. bangii e (6) P. guianensis. Legend: (Starch) double-headed arrow. Scale bars: (1-9) = 20 
887 µm.
888 TABLE
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889 Table 1- Leaf functional traits evaluated in congeneric woody plants in the Rainforest and Amazonian 
890 Savanna physiognomies. Functional traits adapted from De Paula et al. (2019).
891 Table A- Selected congeneric species in the studied phytophysiognomies.
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ed96 et al., 2007). In addition to these characteristics, it presents a hypodermis and investment 
97 in spongy parenchyma, which favors water use efficiency and gas exchange, promoting 
98 higher growth rates (Hoffmann and Franco, 2003; Rossatto et al., 2009a) and 
99 development. In contrast, the phytophysiognomies of Ombrophilous Forests are 
100 characterized by a higher degree of shading and occur in soils with greater availability of 
101 nutrients and water, with a set of leaf functional attributes related to greater water, light, 
102 and nutrient absorption (Hoffmann et al., 2012), favoring an acquisitive strategy in 
103 resource use. Plants have thin leaves, lower growth and specific leaf area rates, as well as 
104 a high degree of stomatal pore opening (Ogburn and Edwards, 2012; Warman et al., 
105 2011). These results demonstrate that differences in soil properties can influence the 
106 functional characteristics of leaves, as well as the distribution arrangement of species in 
107 the Amazon vegetation (Melo-Júnior and Boeger, 2015), which needs further 
108 investigation.
109 Understanding the effects of environmental factors on ecosystem functions 
110 through plant functional composition is essential for predicting the provisioning of 
111 various ecosystem services (Collatz et al., 1991). Currently, the relationships between 
112 historical and ecological determinants have been used in investigations of phylogenetic 
113 approaches to community studies, primarily using congeneric species to avoid functional 
114 responses linked to phylogenetic differences (Elliott et al., 2016; Vamosi et al., 2009). In 
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115 Amazonian forests, the phylogenetic structure of tree communities varies in relation to 
116 soil chemical composition, with low nutrient availability contributing to strong 
117 environmental filtering in savanna and forest areas (Cadote et al., 2020; Cosme et al., 
118 2017; Rossatto et al., 2009b).
119 Recent studies show that the Amazon Domain strongly influences species 
120 composition due to humidity, topography, and the regulation of global climate through 
121 moisture accumulation (Costa et al., 2022; Elias et al., 2019). In tree communities, species 
122 composition is modified by resource availability across environments, among other 
123 factors, which possibly results in niche partitioning and environmental filtering processes 
124 (Bartelheimer et al., 2010; Costa et al., 2005; Rowland et al., 2015). In this context, drier 
125 and hotter environmental and climatic conditions may cause future changes in forest 
126 structure, demography, and functioning, making them more open and particularly 
127 promoting greater plant mortality effects (Allen et al., 2010; Maracahipes et al., 2018). 
128 This study aims to investigate whether there is variation in leaf functional traits among 
129 congeneric species in tree communities under different edaphic conditions in savanna and 
130 forest areas in the Central Amazon. For this purpose, the following hypotheses were 
131 tested: 1 - Contrasting edaphic conditions in savanna and forest areas in the Central 
132 Amazon act as environmental filters modulating leaf functional traits and influencing 
133 species distribution in the environment; 2 - Woody species in the Amazon Savanna 
134 exhibit higher values of leaf functional traits compared to forest trees.
135 The obtained results will be useful in understanding the distribution patterns of 
136 congeneric species along the edaphic gradient in the Amazon Domain, identifying and 
137 selecting leaf characteristics for taxonomic studies, as well as predicting the adaptive 
138 responses of congeneric woody species to climate and environmental changes.
139 2. MATERIAL AND METHODS
140 2.1. Study Area
141 The study was conducted in two sustainable use conservation units in the Central 
142 Amazon, in the Savanna and Ombrophilous Forest formations, in the state of Pará, Brazil. 
143 The Savanna physiognomy is located in the Environmental Protection Area (APA) of 
144 Alter do Chão, municipality of Santarém, at geographic coordinates (2° 29' 27.60" S and 
145 54° 55' 52.96" W), and the Ombrophilous Forest area is located in the municipality of 
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146 Belterra, within the Tapajós National Forest (FNT), at geographic coordinates (2° 29' 
147 27.60" S and 54° 55' 52.96" W) (Fig. 1). The data were collected during the dry season, 
148 in September 2021, and no insects or signs of herbivory were observed on the sampled 
149 leaves.
150 Climatic data on daily average maximum temperature, monthly temperature, and 
151 total precipitation were collected for the 12 months preceding and during the study 
152 (September 2021) from the database of the automatic meteorological station system 
153 available on the website of the Brazilian National Institute of Meteorology (INMET) (Fig. 
154 A). The data were obtained from the Placas A211 station (Fig. A1) and the Santarém 
155 A250 station (Fig. A2), which correspond to the Ombrophilous Forest and Amazon 
156 Savanna physiognomies, respectively.
157 Leaf branches of congeneric woody species were collected in permanent plots 
158 within each vegetation type sampled. For this purpose, two plots were selected in the 
159 PPBio (Biodiversity Research Program) module located at kilometer 117 of BR-163, in 
160 addition to a plot from the LBA project (Large-Scale Biosphere-Atmosphere Experiment 
161 in Amazonia) at kilometer 67 of BR-163, in the Tapajós National Forest (FNT). The 
162 permanent plots in the FNT at kilometers 117 and 67 have dimensions of 40 x 250 m and 
163 50 x 1,000 m, respectively, following the contour lines of the terrain, installed according 
164 to Castilho et al. (2014). For the Alter do Chão APA, four plots with dimensions of 10 x 
165 250 m were selected (Magnusson et al., 2008).
166 The FNT is a sustainable use conservation unit that covers approximately 523,000 
167 hectares and has a hot-humid Ami climate typical of tropical forests (Koppen, 1948), with 
168 an average temperature of 23.5 °C and an annual average precipitation of 2,110 mm, with 
169 more intense periods of rainfall from January to May (Carvalho, 2001; Institute for 
170 Biodiversity Conservation and Monitoring, 2004). Regarding the soils, the region is 
171 predominantly covered by deep Dystrophic Yellow Latosol with varying textures, acidity, 
172 friability, and dense forest cover. The terrain in the region is considered flat to slightly 
173 undulating, with altitudes ranging from 8 to 300 meters above sea level, near bodies of 
174 water (Espírito-Santo et al., 2005; Institute for Biodiversity Conservation and Monitoring, 
175 2004). The Alter do Chão Environmental Protection Area (APA) has an average 
176 temperature of 25.5 °C and an annual average precipitation of 1,907 mm between the 
177 years 1972 and 2022 (National Institute of Meteorology, 2022). The higher regions are 
178 characterized by a mosaic of Igapó and Terra Firme forests formed by the Tapajós River. 
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179 The savanna areas (approximately 10,000 hectares) consist of herbaceous vegetation 
180 composed of grasses, sedges, and eudicotyledons from different botanical families, 
181 distributed in shrub (60-80 cm in height) and tree (up to 10 m in height) strata (Magnusson 
182 et al., 2008b). According to the Koppen classification (1948), the climate of the savanna 
183 region is type Am (tropical climatewith summer rains), and the soils are sandy and acidic, 
184 characterized by low nutrient availability, including calcium and iron, as well as a high 
185 amount of aluminum (Magnusson et al., 2008c; Sarmiento and Monasterio, 2008).
186 2.2. Selection of plant species 
187 The selection of congeneric species in the two studied physiognomies was based 
188 on a floristic inventory by Espírito-Santo et al. (2005) and Magnusson et al. (2008). Nine 
189 arboreal species of congeneric eudicots were selected, with 3 species from the Tachigali 
190 genus and 5 species from the Pouteria genus (Table 1, Fig. 2), which are included in the 
191 most abundant families in the study areas (Fabaceae and Sapotaceae) and have 
192 distributions primarily restricted to one of the extremes of the edaphic gradient (savannas 
193 and forests). For each genus, we paired one species restricted to the savanna physiognomy 
194 (hereafter referred to as savanna species) and another restricted to the forest environment 
195 (hereafter referred to as forest species). Relatively well-resolved taxonomically species 
196 were prioritized based on the literature and expert opinions. Fertile branches were 
197 collected and herbarium specimens were prepared, with the exsiccata deposited in the 
198 Santarém Herbarium (HSTM) at the Federal University of Western Pará, Santarém 
199 campus, Pará, Brazil.
200 2.3. Leaf functional traits
201 2.3.1. Structural and histochemical characterization
202 Leaf functional traits were characterized after collecting completely expanded and 
203 sun-exposed healthy leaves from the third node below the apex of branches of at least 
204 three individuals per species, at the end of the dry season. For trait characterization, four 
205 leaves and three leaflets (central portion of the rachis) were collected for each species of 
206 simple and compound-leaved woody plants, respectively (Table 1).
207 The anatomical characterization of the leaves was performed from the middle 
208 third of the leaf blade. For this, leaf samples were fixed in FAA70 solution (37% 
209 formaldehyde, glacial acetic acid, and 70% ethanol, 1:1:18, v/v/v) (Johansen, 1940) for 
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210 48 hours. Subsequently, leaf fragments were removed from the fixative, immersed in 70% 
211 ethanol preserving solution (Vasconcelos, 2019), and stored in a refrigerator at 5ºC. Later, 
212 the samples were dehydrated in an increasing ethanol series (75, 80, 85, 90, and 95%), 
213 pre-infiltrated, infiltrated, and embedded in histological resin (Leica® historesin), 
214 following the manufacturer's instructions. After embedding, the material was transversely 
215 sectioned (5 µm) using a manual rotary microtome (Crux, EasyPath), stained with 0.05% 
216 toluidine blue in 0.1 M phosphate buffer, pH 6.8 (O'Brien et al., 1964), and mounted in 
217 colorless glass varnish 500â (Paiva et al., 2006). Scientific image recording was 
218 performed using a photomicroscope (Axio Lab.A1, Zeiss microscope coupled with Zeiss 
219 Axiocam ERc 5s digital camera). For histochemical analysis, part of the leaf samples 
220 embedded in resin and transversely sectioned in a microtome were subjected to the 
221 following tests: ferric chloride (Johansen, 1940) to identify general phenolic compounds, 
222 Lugol's reagent (Johansen, 1940) to detect starch, and Sudan IV (Pearse, 1972) to evaluate 
223 structural lipids and cuticle. 
224 For the structural characterization and micromorphometry of the leaves, the 
225 analysis of functional attributes (qualitative and quantitative) with functional significance 
226 (Table 1) was performed. Qualitative descriptions followed the references established in 
227 De Paula et al. (2019). Quantification of leaf functional attributes (internal and external 
228 morphology) was performed with 60 measurements using a 40x objective lens tissue 
229 sample per individual, and then the average value per individual was calculated. These 
230 measurements were performed using the ImageJ software (Abràmoff et al., 2004).
231 2.3.2. Stomatal density and index
232 For hydraulic characterization and leaf surface analysis, the epidermis separation 
233 method was used, where leaf portions were placed in tubes containing a 1.5% active 
234 chlorine bleach solution in a 1:1 v/v ratio, for approximately 60 hours. After this period, 
235 the samples were washed in distilled water, followed by separation of the adaxial and 
236 abaxial epidermal surfaces with the aid of a brush, staining with 0.1% basic fuchsin 
237 (Roesse, 1962), and mounting in a semipermanent medium of glycerinated gelatin (Kraus 
238 and Arduin, 1997).
239 The hydraulic attributes (stomatal density and index) on the leaves (Table 1) were 
240 measured after epidermal dissociation. Stomatal counts were made in 10 randomly chosen 
241 microscopic fields for each epidermal surface and individual studied. Each field consisted 
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242 of 0.06 mm² with a 40x magnification. A total of 100 fields were used to determine 
243 stomatal frequency or index (Salisbury, 1927). The number of stomata per field was 
244 converted to the number of stomata mm-2. The stomatal index (SI) was estimated as (S/(E 
245 + S)) × 100, where S is the number of stomata and E is the number of epidermal cells per 
246 unit leaf area (Pompelli et al., 2010).).
247 2.4. Soil physicochemical analysis
248 For the physicochemical analysis of the soil, 6 soil samples were collected in each 
249 plot using a Dutch auger, at 4 different depths: 0-5 cm, 5-10 cm, 10-20 cm, and 20-30 
250 cm. A composite sample was prepared for each depth, following the protocol of Barbosa 
251 et al. (2006). Subsequently, the samples were sent to the Soil Department's routine 
252 laboratory at the Federal University of Viçosa, Minas Gerais, where the following 
253 parameters were determined: pH, P, K, Ca+2, Mg+2, Na+, Al+3, Fe, Zn, Cu, Mn, S, B, and 
254 soil texture (percentage of clay, silt, and sand), according to the methods adopted by 
255 Tedesco et al. (1997). To reduce the number of soil variables, only the soil variables from 
256 the 5-10 cm depth were considered in the statistical analyses, as they are less sensitive to 
257 short-term changes caused by vegetation (Gray and Bond, 2015).
258 2.5. Data analysis
259 To analyze whether species of the same genus occurring in contrasting soil 
260 conditions invest differently in leaf functional attributes between savanna and rainforest 
261 environments, paired t-tests were used with mean values of species characteristics. A 
262 significance level of 0.05 was used for all analyses. All variables were checked for 
263 normality and homoscedasticity using the Shapiro-Wilk and Levene tests (Levene, 1961; 
264 Shapiro and Wilk, 1965). Phylogenetic relationships among the studied species were 
265 reconstructed using PhyloMatic v.3 (Webb & Donoghue, 2005), based on the maximum 
266 resolution of the angiosperm phylogeny (R20120829 tree). Phylogenetic trees were 
267 constructed in Phylocom 4.2 and then used to calculate phylogenetically independent 
268 contrasts with the R package.
269 A principal component analysis (PCA) was used to evaluate the patterns of 
270 covariation among the characteristics (all attributes evaluated in this study) and to 
271 describe species strategies in different vegetation types. Additionally, PCA was used to 
272 analyze the multivariate associations of leaf functional attributes and their relationship 
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273 with vegetation types. All statistical analyses were performed using R software (R Core 
274 Team, 2022).
275 3. RESULTS
276 3.1. Leaf Anatomy
277 In the genus Tachigali, in transverse view of the leaf blade (Fig. 3 A-H), the 
278 species have uniseriate epidermis (cells with straight walls) on both leaf surfaces and a 
279 heterogeneous dorsiventral mesophyll type (with one layer of elongated palisade 
280 parenchyma and four to five layers of spongy parenchyma, occupying over 50% of the 
281 mesophyll) with crystalline inclusions, regardless of the vegetation type (Fig. 3 D-G). In 
282 the forest environment, leaves with tector trichomes and a collateral bundle in the midrib 
283 were observed visually. In the savanna species, differences in the structure of the midrib 
284 with a bifurcated collateral bundle were apparently observed (Fig. 3 C-F).
285
286 In the frontal view, the leaf surfaces of species belonging to the genus Tachigali 
287 have sinuous and thick-walled epidermal cells, and they are hypostomatic, regardless of 
288 the studied phytogeomorphies (Fig. B 1-8). In the forest area, the presence of tector 
289 trichomes on the abaxial surface of the leaf of T. chrysophylla was observed (Fig. B 4), 
290 while the other studied species were glabrous (Fig. B 5 and 7). In this phytogeomorphy, 
291 the stomata of T. alba (abaxial surface of the leaf) are paracytic (Fig. B 6). In the savanna 
292 environment, a large number of anomocytic stomata were observed on the abaxial surface 
293 of the leaf of T. vulgaris, arranged above the level of the other epidermal cells (Fig. B 8).
294 In the genus Pouteria, in the cross-section of the leaf blade (Fig. 4 A-L), the 
295 evaluated species have a thick uniseriate epidermis (cells with straight walls) on both leaf 
296 surfaces, heterogenous dorsiventral mesophyll (with a layer of elongated palisade 
297 parenchyma and six to eight layers of spongy parenchyma, occupying more than 50% of 
298 the mesophyll), glabrous leaves, and visually prominent intercellular spaces in the spongy 
299 parenchyma (Fig. 4 D-L), regardless of the phytogeomorphies (Fig. 4 A-I). In the forest 
300 area, leaves with less thick cuticle compared to savanna species were observed (Fig. 6 B), 
301 and crystal inclusions of the prismatic type (Fig. 4 G, I). Differences in the structure of 
302 the main vein were observed in the savanna species, showing higher amounts of tannin 
303 idioblast inclusions (Figs. 4 C and E).
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304 In the frontal view, species belonging to the genus Pouteria exhibited 
305 hypostomatic leaves in the different studied areas (Fig. C 4-6, 10-12). In the 
306 Ombrophilous Forest phytogeomorphy, the epidermal cells are sinuous and thick 
307 (especially on the adaxial surface) and have paracytic stomata in P. procera, P. reticulata, 
308 P. baehniana (Fig. C A-L), and anomocytic stomata in P. bangii (Fig. C 16, 17). In the 
309 Savanna, the species have thick epidermal cells with straight walls and generally 
310 rectangular shape, along with paracytic stomata in P. ramiflora and P. guianensis (Fig. C 
311 3, 8, 11, 16, 17).
312 3.2. Histochemistry
313 Histochemical tests for non-structural phenolic compounds indicated that the 
314 species Tachigali vulgaris from the savanna area has a higher visual amount of this 
315 compound in its leaves compared to the other woody species from the forest area (Fig. D 
316 1-3). In both studied phytogeomorphies, congenetic species of Tachigali showed lipids 
317 in the chemical composition of the cuticle on the adaxial surface of the leaf, with emphasis 
318 on T. chrysophylla in the forest area (Fig. D 4-6). Histochemical tests indicated a higher 
319 visual amount of starch in the leaves of T. chrysophylla, predominantly in the palisade 
320 and spongy parenchyma, compared to the other studied species (Fig. D 7-9). 
321 Histochemical tests for non-structural phenolic compounds in the leaves of 
322 congenetic woody species of Pouteria indicated that the species P. ramiflora and P. 
323 guianensis from the savanna area have a higher visual amount of these compounds in the 
324 mesophyll, compared to the forest species studied (Fig. E 1-6). On the adaxial surface of 
325 the leaf, regardless of the woody species and sampled phytogeomorphy, the cuticle is 
326 well-defined and composed of lipids, with emphasis on P. reticulata, P. baehniana, and 
327 P. guianensis (Fig. F 1-6). Regarding the presence of carbohydrates in the leaf blades of 
328 Pouteria, qualitative tests indicated a higher content of starch in the mesophyll (palisade 
329 and spongy parenchyma) of P. procera and P. reticulata, both woody species from forest 
330 areas (Fig. G 1-6).
331 3.3. Density and stomatal index
332 Regarding the measured hydraulic attributes (density - STD and stomatal index - 
333 STI) in 10 congenetic pairs evaluated, 8 species from the savanna phytogeomorphy 
334 exhibited higher values of STD and STI compared to the forest species, contrary to what 
335 was observed for the remaining evaluated pairs (Fig. 5 A-B). A paired t-test between 
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336 phylogenetic pairs indicated that the STD of the forest species was lower than that of the 
337 congenetic savanna species (paired t-test t = 4.2; df = 8; P = 0.0012; Fig. 5A), indicating 
338 a significant differentiation of this hydraulic attribute in relation to the environment 
339 regardless of the genus. However, the pairs of species P. procera and P. ramiflora, and 
340 P. bangii and P. guianensis, highlighted in yellow and orange, respectively, exhibited 
341 values contrary to those found for this attribute (Fig. 5 A, B).
342 In relation to the stomatal index (STI) evaluated in the leaves of congenetic trees, 
343 the paired t-test between phylogenetic pairs indicated significant variation in the values 
344 found (paired t-test t = 3.1; df = 7; P = 0.001; Fig. 5B) for the studied species. The highest 
345 STI values were found in the savanna environment compared to the forest species, 
346 indicating that regardless of the genus, there is variation in this attribute along the edaphic 
347 gradient (Fig. 5A-B). However, the pairs of P. procera and P. ramiflora, and P. bangii 
348 and P. guianensis, highlighted in yellow and orange, respectively, showed inverse values 
349 compared to those found for this attribute (Fig. 5B).
350 3.4. Internal and external leaf morphology
351 Significant variations in leaf tissue thickness (leaf blade, adaxial surface cuticle, 
352 epidermis on both surfaces, and palisade and spongy parenchyma) were found among the 
353 different studied phytophysiognomies in the congenetic species (Figs. 6 A-F). The 
354 savanna species showed greater leaf and cuticle thickness compared to the woody plants 
355 from the forest environment (For leaf thickness, the paired t-test was t = 5; df = 9; P = 
356 0.001; Fig. 6A. And for cuticle thickness, the paired t-test was t = 3.5; df = 8; P = 0.001; 
357 Fig. 6B). In both studied phytophysiognomies, the attributes of leaf thickness and cuticle 
358 thickness in the congenetic pair of P. procera and P. ramiflora showed the least variation 
359 among the different phytophysiognomies, contrary to what was observed for the T. alba 
360 and T. vulgaris pair (Fig. 6C).
361 The congenetic species sampled in the savanna environment also showed the 
362 highest values of epidermal thickness on the adaxial and abaxial surfaces of the leaf, 
363 compared to the other forest species (Fig. 6C, D), respectively. A significant difference 
364 in the thickness of the palisadeparenchyma (paired t-test t = 4.2; df = 8; P = 0.001; Fig. 
365 6F) and spongy parenchyma (Table 1; paired t-test t = 4.2; df = 8; P = 0.001; Fig. 6E) was 
366 found in the mesophyll in the two studied phytophysiognomies (Fig. 6E, F). The woody 
367 plants from the savanna area had a greater thickness of palisade parenchyma compared to 
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368 the forest environment, except for the P. procera and P. guianensis pair, which showed a 
369 reduction in values for this attribute (Fig. 6F). Regarding the thickness of the spongy 
370 parenchyma, the congenetic species from the forest phytophysiognomy showed the 
371 highest values for this attribute compared to the savanna area (paired t-test t = 3.2; df = 
372 7; P = 0.001; Fig. 6E), except for the P. reticulata and P. ramiflora pair, which maintained 
373 constant values, and the T. alba and T. vulgaris pair, which showed a lower value in the 
374 forest area (Fig. 6E).
375 In general, Principal Component Analysis (PCA) of functional leaf traits and the 
376 types of phytophysiognomies showed segregation between forest and savanna species for 
377 the genera Tachigali and Pouteria (Fig. 7). The functional characteristics of species 
378 occurring in different areas were primarily separated based on water storage capacity, 
379 protection, and drought resistance. The first two axes of the PCA explained 69.8% of the 
380 total data variation, with axis 1 and axis 2 representing 40.9% and 28.9%, respectively 
381 (Fig. 7). 
382 In the PCA of soil physicochemical analysis in different phytophysiognomies, it 
383 was observed that the two main axes accounted for 87.8% of the total data variation, with 
384 axis 1 and axis 2 representing 70.8% and 17.0%, respectively (Fig. 8). The PCA indicated 
385 that there is a pattern of convergence between the forest plots and savanna plots. 
386 However, there was a divergence between the two environments in terms of soil chemical 
387 analysis, with pH, Ca, and Silt being more correlated with the savanna plots, while Na, 
388 Fe, K, Al, organic matter, P, Mg, N, and Clay were more correlated with the forest plots 
389 (Fig. 8).
390 4. DISCUSSION
391 The data obtained in the present study indicated that congeneric species of 
392 Tachigali and Pouteria occurring in two phytophysiognomies in the Amazon Domain 
393 with contrasting nutrient availability exhibit different arrangements of functional leaf 
394 morphophysiological traits. These results support the first hypothesis proposed here, that 
395 edaphic conditions act as environmental filters, which modulate leaf functional 
396 adjustments and influence the distribution of woody species at a local scale. Additionally, 
397 in tree species of the Amazonian Savanna, higher values of functional leaf traits were 
398 found compared to those of the Forest, corroborating the second hypothesis proposed 
399 here. The use of qualitative and quantitative analyses in the present study indicated that 
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400 species present in the savanna environment, with lower nutrient availability, exhibited 
401 more conservative traits related to water storage and drought resistance. However, for 
402 species in the forest area, which generally have a higher nutrient availability in the 
403 environment, more acquisitive strategies were observed, consistent with Araújo et al. 
404 (2021) and Ariano and Silva (2016) who also worked with other woody species in the 
405 Amazon Domain. 
406 In this context, stomatal density (STD) was higher in the savanna 
407 phytophysiognomy compared to the forest species. Overall, an increase in STD is a 
408 compensatory factor for CO2 uptake and the maintenance of photosynthetic and 
409 respiratory activity. Plants from savanna environments tend to have smaller stomata to 
410 achieve rapid stomatal opening and closing responses, in order to minimize water loss 
411 through evapotranspiration (Pireda et al., 2020; Ariano, 2022). The data found here 
412 contradicts the findings reported by Pearce et al. (2006) and Rossatto et al. (2009c) who 
413 worked with different groups but in the same phytophysiognomy, indicating high STD 
414 rates in xeric environments, where plants have lower water and/or nutritional availability 
415 and higher light availability. 
416 The stomatal index (STI) showed the same trend as STD, being higher in savanna 
417 species compared to forest species. These results differ from those found by Durand et al. 
418 (2019), who observed lower STI values for these species in the forest phytophysiognomy. 
419 Generally, a lower STI is expected in savannas, as plants in this environment have 
420 substantially smaller adjacent epidermal cells, aiming to promote faster stomatal closure 
421 in case of increased temperature, light availability, and reduced water availability 
422 (Durand et al., 2019b). Thus, the results of STD and STI for the species of the different 
423 genera evaluated here show that the responses of these traits are not directly related to 
424 environmental conditions but are specific to the studied genera.
425 Regarding leaf blade, woody species present in the Amazonian Savanna exhibited 
426 the thickest leaf, cuticle, epidermis (both surfaces), and palisade parenchyma, compared 
427 to those in the Ombrophilous Forest, regardless of the genera. The structural variations 
428 displayed by the cuticle and epidermis are adaptive strategies of species related to xeric 
429 environments and serve to reduce the loss of water vapor from the internal leaf tissues to 
430 the atmosphere, improving the efficiency of water use (Lambers and Oliveira, 2019; 
431 Larcher and Prado, 2000). Perhaps this characteristic is related to genetic inheritances 
432 from biogeographic processes that go beyond our analyses (Ariano, 2022). 
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433 The thickening of the epidermis in plants from savanna environments is an 
434 adaptive protective characteristic that provides greater support against radiation, as well 
435 as water conservation and photosynthetic efficiency, which can be mainly regulated by 
436 the abaxial leaf surface, ensuring the arrival of light in adequate quantity and quality for 
437 photosynthesis to occur, avoiding, for example, a process of photoinhibition (Taiz and 
438 Zeiger, 2006; Maréchaux et al., 2020). 
439 The greater thickness of the palisade parenchyma was found in the savanna 
440 environment and is more related to the higher irradiance found in this environment 
441 (Ariano and Silva, 2016; Simioni et al., 2017). Several authors have stated that the thick 
442 cuticle, epidermis (adaxial leaf surface), and palisade parenchyma are plastic attributes 
443 that can differ among communities, populations, and genera that colonize locations with 
444 different environmental pressures, including the edaphic gradient (Araújo et al., 2021e; 
445 Ariano, 2022; Liu et al., 2019; Rossatto et al., 2010, 2009b). In addition, anatomical 
446 characteristics such as the thickness of the spongy parenchyma in the leaves of congeneric 
447 species differed in the present study, regardless of the environment, which may confer 
448 greater adaptive capacity of these species to different future climatic, environmental, and 
449 edaphic patterns (Araújo et al., 2022; Àvila-Lovera et al., 2022).
450 Histochemical analysis highlighted the presence of starch in the leaves of 
451 Tachigali and Pouteria woody species, with its highest accumulation apparently452 occurring in the forest area. In this context, in environments with greater nutrient 
453 availability, such as the ombrophilous forest areas, plants exhibit acquisitive strategies 
454 and accumulate more carbohydrates than plant species from other phytophysiognomies, 
455 thus ensuring greater plant survival in this environment (Pessoa et al., 2021). The 
456 presence of phenolic compounds was highly evident in savanna species, both in Tachigali 
457 and Pouteria. The accumulation of these compounds strategically provides an ecological 
458 advantage for savanna plants and has a protective function against increased exposure to 
459 solar radiation and herbivory, as well as abiotic stresses (e.g., drought, heat, nutrient 
460 availability), and modulation of ecosystem processes through effects on decomposition 
461 and nutrient cycling that affect plant communities and food webs (Westoby et al., 2002, 
462 Hunter, 2016)). 
463 In the PCA, there was a clear distinction between the Ombrophilous Forest and 
464 Amazonian Savanna phytophysiognomies along the edaphic gradient, driven mainly by 
465 differences in leaf morphoanatomy (Fig. 7 and 8). As this gradient represents different 
466 nutrient and water availabilities for tree species, the plasticity of leaf functional traits can 
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467 be essential for plants to adapt to environmental changes (Viani et al., 2014c; Yu et al., 
468 2019). The PCA confirmed previous findings in the micromorphometric analyses, 
469 indicating that savanna exhibits higher values of leaf functional traits compared to the 
470 forest. These data support findings by Simioni et al. (2017), who demonstrated that the 
471 mentioned leaf functional traits are likely shaped by environmental determinism.
472 The analysis of the physicochemical soil composition in the study areas indicated 
473 variation in the morphoanatomical functional leaf characteristics along the edaphic 
474 gradient, explaining a possible organization of the plant community (Fig. 8). Forest plots 
475 showed a higher correlation with fertility, evidenced by higher concentrations of 
476 macronutrients such as high levels of organic matter, N, Na, Al, Fe, K, P, Mg, coarse and 
477 fine sand, and silt and clay. In the savanna environment, the plots were more correlated 
478 with pH, Ca, and silt, suggesting lower nutrient availability. These findings support our 
479 hypothesis that the variation in leaf functional traits may be related to the gradient, due to 
480 more severe environmental filters in the savanna area, mainly related to soil nutrients and 
481 water deficit (Hoffmann et al., 2005; Viani et al., 2014).
482 It is predicted that climate and environmental changes will increase extreme 
483 weather events and generate changes in forest structure, demography, and functioning, 
484 making them more open and promoting greater species tree mortality effects 
485 (Maracahipes et al., 2018; Yu et al., 2019). Therefore, studies on species that are already 
486 experiencing critical levels of temperature increase and low nutrient availability are 
487 essential for understanding the potential behaviors of habitats and the assembly of 
488 communities under future climate change. Focusing on plant functional traits is 
489 interesting because it allows us to understand the complex dynamics of the community 
490 and predict the effects of ongoing environmental changes (Schrodt et al., 2015).
491 Understanding the drivers of adaptive characteristics, as well as those at the 
492 physiological and molecular levels, of the Ombrophilous Forest and Amazonian Savanna 
493 phytophysiognomies represents a fundamental step in applying trait-based approaches to 
494 the ecology of tree species communities, as the physical environment can strongly 
495 influence ecosystem function variation (Bu et al., 2019; Liu et al., 2021). Our results 
496 suggest that the phenotypic plasticity of leaf functional traits, particularly leaf thickness, 
497 cuticle, epidermis (both surfaces), palisade parenchyma, and soil physicochemical 
498 composition (STD, STI), contribute to the growth and establishment of Tachigali and 
499 Pouteria plant species along the edaphic gradient in the Amazon Domain. 
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500 5. CONCLUSION
501 The structural and hydraulic functional traits in Amazonian tropical woody 
502 species at a local scale, across environments with contrasting edaphic conditions, showed 
503 continuous variation in leaf functional attributes within the two studied 
504 phytophysiognomies. The morphofunctional traits for both genera may be responses to 
505 low soil nutrient content and/or water scarcity and high solar radiation. Some species 
506 responded to the environment with greater investment in cuticle, adaxial and abaxial 
507 epidermis, stomatal density, and stomatal index. Forest species responded to the 
508 environment with increased investment in traits that reduce water loss to the environment, 
509 while savanna woody species exhibited a higher amount of palisade parenchyma, 
510 promoting greater water and nutrient retention within the leaf, as well as improving 
511 photosynthetic performance. The attributes most related to conservative strategies were 
512 cuticle thickness, adaxial and abaxial epidermis, palisade parenchyma thickness, and 
513 stomatal density and index. Attributes related to acquisitive strategies were lacunary 
514 parenchyma thickness and leaf thickness. 
515 The variations in edaphic conditions between phytophysiognomies (Savanna and 
516 Forest) appear to be strong environmental filters that drive morphophysiological 
517 adjustments in leaves, which may explain species distribution in the environment. In a 
518 scenario of climate and environmental changes, understanding the responses of leaf 
519 functional traits to edaphic conditions can predict the distribution and future maintenance 
520 of species in the Amazon Domain.
521
522 Consent to Participate
523 All authors agree to participate in this publication.
524 Author Credit Statement
525 Lucas Vinicius Cavalcante Esteves: Methodology, Formal Analysis, 
526 Visualization, Writing - Original Draft, Writing - Review and Editing. Kleyton Kleber 
527 dos Santos Correia: Assistance in statistical analysis. Mayda Cecília dos Santos 
528 Rocha: Data curation. Gleicy Assunção Rodrigues: Data curation. Darlisson Mesquita 
529 Batista: Assistance in phylogenetic analysis and data curation. Anderson Alves-Araújo: 
530 Plant species analysis and identification. Marina Magalhães Hirota: Funding 
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531 acquisition, Resource acquisition, Project administration. Deliane Vieira Penha: 
532 Investigation, Writing - Review and Editing. Leandro Lacerda Giacomin: 
533 Conceptualization, Investigation, Data curation, Writing - Review and Editing. Advanio 
534 Inácio Siqueira-Silva: Funding acquisition and resources, Project administration, 
535 Supervision, Conceptualization, Methodology, Visualization, Writing - Original Draft, 
536 Writing - Review and Editing.
537 Author Credit Statement
538 The authors declare that they have no known competing financial interests or 
539 personal relationships that could have influenced the work reported in this article.
540 Author Credit Statement
541 This study was funded by the PELD-POPA Project, the Graduate Program in 
542 Biodiversity of the Federal University of West Pará (Ufopa) through Academic Support 
543 Development,Instituto Serrapilheira (grant number Serra-1709-18983), and 
544 BCB/ICTA/Ufopa.
545 Author Credit Statement
546 The authors would like to thank Instituto Serrapilheira, PELD-POPA, the Botany 
547 Laboratory (LaBot - BCB/ICTA/Ufopa), the Multi-User Laboratory of Multifunctional 
548 Analysis (Labam- BEP/ICTA/Ufopa), and the Biology Teaching Laboratory II 
549 (ICED/Ufopa).
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797 TABLE
798 Table 1- Leaf functional traits evaluated in congeneric woody plants in the Rainforest and Amazonian 
799 Savanna physiognomies. Functional traits adapted from De Paula et al. (2019).
Attributes Main Functional Significance
Cuticle thickness Foliar protection; water conservation; radiation reflection
Epidermis thickness Foliar protection/support; water conservation; light reflection
Trichomes Foliar protection; water balance; radiation reflection
Subepidermal layers Foliar support; water conservation; radiation reflection
Palisade parenchyma thickness Carbon gain; protection against excessive radiation
Leaf thickness Resource acquisition and utilization; resistance to physical damage
Spongy parenchyma thickness Carbon gain
Mesophyll organization Carbon gain; water conservation
Presence of sclerenchyma Mechanical support; leaf defense
Presence of stomata Carbon gain; water conservation
Stomatal density Carbon assimilation and water use efficiency
Stomatal index Carbon assimilation and water use efficiency
800
801 Table A- Selected congeneric species in the studied phytophysiognomies.
Species Family Phytophysiognomy Accession Number
Tachigali chrysophylla (Poepp.) Zarucchi & Herend. Fabaceae Forest HSTM 15338
Tachigali alba Ducke. Fabaceae Forest HSTM 15342
Tachigali vulgaris L.G.Silva & H.C.Lima. Fabaceae Savanna HSTM 13173
Pouteria bangii (Rusby) T.D.Penn. Sapotaceae Forest HSTM 15386
Pouteria procera (Mart.) K.Hammer. Sapotaceae Forest HSTM 15385
Pouteria baehniana Monach. Sapotaceae Forest HSTM 15384
Pouteria reticulata (Engl.) Eyma. Sapotaceae Forest HSTM 15383
Pouteria guianensis Aubl. Sapotaceae Savanna HSTM 15389
Pouteria ramiflora (Mart.) Radlk. Sapotaceae Savanna HSTM 15387
802
803
804
805 FIGURES
806 Fig. 1. Map of the study area. The highlighted areas in black and pink represent the Ombrophilous Forest 
807 physiognomy, and the blue color represents the Savanna area, located in the western state of Pará, Brazil. 
808 Fig. 2. Evolutionary relationship of 9 tree species belonging to the Rainforest and Amazonian Savanna 
809 physiognomies. The cladogram was based on the phylogeny of well-resolved angiosperms (tree 
810 R20120829). The colors indicate the environment to which the species belong (blue for forest, yellow for 
811 savanna). 
812 Fig. 3. Anatomical characterization of the leaf of species belonging to the genus Tachigali (cross-section) 
813 in different phytogeomorphies (Forest and Savanna) in Central Amazon. (A, D, E) Tachigali chrysophylla, 
814 (B, F) T. alba, and (C, G, H) T. vulgaris. A-C: Midrib. D, F, G: Leaf blade. E, H: Detail of crystalline 
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815 inclusions in T. chrysophylla and T. vulgaris, respectively. Legend: (Ead) adaxial epidermis, (Eab) abaxial 
816 epidermis, (arrow) Cuticle, (S) Stomata, (X) Xylem, (Hip) Hypodermis, (Pp) Palisade parenchyma, (Pl) 
817 Spongy parenchyma, (Fv) Vascular bundle, (FF) Bundle fibers, (double arrowheads) Tannin idioblast. 
818 Scale bars: (A-D, F, G) = 20 µm and (E, H) = 10 µm.
819 Fig. 4. Anatomical characterization of the leaf of species belonging to the genus Pouteria (cross-section) 
820 in different phytogeomorphies (Forest and Savanna) in Central Amazon. A-C: Midrib. D-I: Leaf blade. (A, 
821 D, E) Pouteria procera, (B, F, G) P. reticulata, (I, L, H) P. baehniana, (J, N, O) P. bangii, (C, H) P. 
822 ramiflora, and (K, P, Q) P. guianensis. A-C, I-K: Adaxial surface of the leaf. D-H, L: Abaxial surface of 
823 the leaf. E-Q: Details of tannin idioblasts and prismatic