Occurrence of homobaric and heterobaric leaves in two forest types of southern Brazil

In ombrophilous forests, light stratifi cation provokes diff erent adjustments by plants for better use of the environmental conditions of each stratum. Among the morphological traits that vary with strata, the presence of bundle sheath extensions (BSEs) is related to water transport, photosynthesis, and leaf mechanical support and classifi es leaves as homobaric or heterobaric. Th is study analyzed the proportion of these types of leaves in a Lowland Ombrophilous Dense Forest (LLODF) and a Mixed Ombrophilous Forest (MOF), and among the strata of each forest type. Th e morphological leaf traits of 89 LLODF tree species and 57 MOF tree species were examined. Th e proportion of homobaric and heterobaric leaves did not diff er between forests. However, in both forest types, the distribution of species with heterobaric or homobaric leaves depended on strata, with heterobaric species occurring mainly in higher strata, and homobaric species in lower strata. Th us, light stratifi cation acts as an ecological fi lter on the composition of the vegetation of these forests, favoring heterobaric species in places with higher light intensity and temperature, such as the highest strata of canopy. On the other hand, homobaric species are more frequent in lower strata, where light is less available and humidity higher.


Introduction
Environmental factors aff ect the growth and survival of plants (Valladares & Niinemets 2008) and infl uence their internal organization (Dickson 2000).In forests, variation in abiotic features along vertical stratifi cation provokes diff erent adjustments by plants for better use of the environmental conditions of each stratum (Valladares & Niinemets 2008;Niinemets 2010;Inoue et al. 2015).Such adjustments can be morphological, physiological, and/or phenological.Among such morphological traits, the presence of bundle sheath extensions (BSEs) is related to water transport (Zwieniecki et al. 2007), photosynthesis (Pieruschka et al. 2010), and leaf mechanical support (Turner 1994).
Bundle-sheath extensions (BSEs) are formed by parenchyma or sclerenchyma cells that extend from the vascular bundle to both sides of the leaf epidermis (Karabourniotis et al. 2000;Nikolopoulos et al. 2002).Leaves are classifi ed as homobaric or heterobaric depending on the condition of the BSEs (Kenzo et al. 2007).Th e former lack, or have incomplete, BSEs and their mesophyll is more homogeneous.The latter have complete BSEs and their mesophyll is divided into several photosynthetic compartments (Karabourniotis et al. 2000;Nikolopoulos et al. 2002;Kenzo et al. 2007).Homobaric leaves have a continuous mesophyll (Terashima 1992).
Such structural differences are reflected in the functional proprieties of these leaf types (Kenzo et al. 2007;Pieruschka et al. 2010;Lynch et al. 2012;Inoue et al. 2015).The presence of BSEs can protect leaf lamina from hydric stress and increase light absorption and mechanical support (Terashima 1992;Karabourniotis 1998;Nikolopoulos et al. 2002;Rhizopoulou & Psaras 2003).Yet in homobaric leaves, gas diffusion in the mesophyll can be more efficient due the absence of BSEs (Pieruschka et al. 2006).Also, BSEs have been linked to light distribution within the mesophyll, allowing investments in thicker and, consequently, smaller leaves (Nikolopoulos et al. 2002).
Despite the light heterogeneity that characterizes Brazilian forests, the occurrence of heterobaric leaves in many plant formations of these biomes and their relation to light gradients is still poorly studied.This study reports on the morpho-anatomical traits of tree species from a Lowland Ombrophilous Dense Forest and a Mixed Ombrophilous Forest in order to investigate the presence and proportion of homobaric and heterobaric leaves in both forest types.Our hypotheses are: a) the frequency of heterobaric leaves is similar in the two studied forest types since both experience similar environmental conditions (annual precipitation and light stratification), independent of their floral composition; b) the distribution and frequency of homobaric and heterobaric leaves vary among different forest strata in response to light stratification, with heterobaric leaves occurring mainly in upper strata and c) leaf type is dependent upon micro-environmental features more so than taxonomic group, as represented by families and/or genus.

Materials and methods
This study was based on leaf morphological data collected during previous studies in two forest sites: a Lowland Ombrophilous Dense Forest (LLODF), located at Volta Velha Reserve (26º04'S, 48º38'W), within the city of Itapoá, SC (a detailed description can be found in Boeger et al. 2004); and a Mixed Ombrophilous Forest (MOF), located in the Botanical Garden Francisca Maria Garfunkel Rischbieter (25º23'10"S, 49º12'58''W), within the boundaries of the city of Curitiba, PR (for more details, see Silveira et al. 2015).The environmental characteristics of each forest type are summarized in Tab. 1.All species included in this study were selected according to two criteria: 1) higher values of importance based on a previous phytosociological survey and 2) the presence of at least three individuals in the forest type.All specimens of collected from LLODF were deposited in UPCB (Herbarium of Department de Botany, UFPR, Curitiba, PR) and specimens from MOF were deposited in MBM (Herbarium of Municipal Botanical Museum, Curitiba, PR).
Morphological data, such as leaf area, leaf thickness, and the presence of homobaric and heterobaric leaves, were collected from 89 LLODF (Tab.2) and 57 MOF tree species (Tab.3).Leaves with complete BSEs were classified as heterobaric while leaves with incomplete and/or no BSEs were classified as homobaric leaves, according to Kenzo et al. (2007).The nomenclature and taxonomic classification of each species were checked against International Plant Names Index (www.ipni.org).
The mean values for the distinct strata of each forest type were compared through One-way ANOVA followed by Tukey test.Leaf area and leaf thickness of homobaric and
The distribution of species with heterobaric leaves among forest strata differed significantly in LLODF (χ2 test, P=0.013, DF=3, N=89 species).The highest proportion of heterobaric leaves was found in Stratum 4 (Tab.4), and the number of heterobaric leaves was directly proportional to light stratification.In MOF, despite the fact that heterobaric leaves were not significantly distributed among strata (χ2 test, P=0.0662, DF=3, N=57 species), an increase in heterobaric leaves was directly related to light intensity, with the highest proportion of heterobaric of leaves being in Stratum 3, since the frequency of heterobaric leaves was 0% in Stratum 4, which was composed of a single species (A.angustifolia) with only homobaric leaves.The lower strata had higher proportions of homobaric leaves in both forests (Tab.4).
For all species considered in LLODF, leaf area did not differ among Strata 1, 2, and 3, but was higher in Stratum 4 (Tab.5).When we excluded species with leaf area > 100 cm 2 , [Ormosia arborea in Stratum 1; Aparisthmium cordatum and Cupania oblongifolia in Stratum 2; Miconia cabucu in Stratum 3 and Coccoloba warmingii in Stratum 4], the average leaf area of Stratum 4 differed from Strata 2 and 3.Although these species occur in small numbers in each stratum, they significantly affected average leaf area, as shown by the standard deviations (Tab.5).In MOF, there was no variation in leaf area among lower strata; only Stratum 4 differed due to the reduced leaf area of A. angustifolia leaves (Tab.5).
In LLODF, leaf thickness did not exhibit the same pattern of variation among strata as leaf area.Only Stratum 3 differed by having thinner leaves than the other strata.When we excluded the species with leaf area > 100 cm 2 , mean leaf thickness varied as follows: Stratum 1 = Stratum 2 > Stratum 3 > Stratum 4. In MOF, Stratum 4 had thicker leaves than the other strata (Tab.5).
The distribution of heterobaric leaves among some plant families was also evaluated (χ 2 test, P < 0.0001, GL = 7, N = 81 species from eight families with more than five species, Tab.S01 in supplementary material).In this study, all species of Lauraceae had heterobaric leaves, independently of strata (Fig. 2), except Endlicheria paniculata, which had heterobaric   Occurrence of homobaric and heterobaric leaves in two forest types of southern Brazil leaves in Stratum 1 at LLODF.Besides Lauraceae, Euphorbiaceae, Fabaceae, Myrtaceae, Primulaceae, and Sapotaceae also had species with both leaf types, with heterobaric leaves being mainly distributed in Strata 2 and 3.No species of Aquifoliaceae and Melastomataceae, which occurred mainly in Strata 1 and 2, had heterobaric leaves.

Discussion
Both forest types exhibited similar proportions of species with homobaric and heterobaric leaves, with a greater occurrence of the former.Heterobaric leaves are generally associated with deciduous forests with cold or dry, well-defined seasons (Terashima 1992).On the other hand, homobaric leaves occur in evergreen forests, generally found in humid and hot regions (Kenzo et al. 2007).Thus, our data corroborate a previous study that found a higher proportion of homobaric leaves in humid forests with high precipitation throughout the year (Kenzo et al. 2007), such as our study sites.Although heterobaric leaves are associated with drier, deciduous forests, they are also present in humid forests, such as was found in our study sites.The distribution of these two types of leaves among strata in the present study was similar to that observed by Kenzo et al. (2007) in a rain forest at Sarawak, Malasia.
The distribution analysis indicated that heterobaric leaves are more common in Strata 3 and 4 in MOF and LLODF, respectively, while homobaric leaves were more common in Strata 1 and 2. This distribution of homobaric and heterobaric leaves in different strata appears to be due to micro-environmental gradients associated with the various forest strata (Kenzo et al. 2007).Such gradients include light availability, temperature, vapor pressure deficit, and wind (Théry 2001;Kitajima & Poorter 2010;Bennett et al. 2015;Inoue et al. 2015).Of these, light availability is particularly important because it can influence the growth, survival, and subsequent reproduction of young individuals (Chazdon et al. 1996;Valladares & Niinemets 2008).
In the canopy, for example, plants are subjected to intense light and heat, which can be stressful during some periods of the day and/or year (Théry 2001;Valladares & Niinemets 2008).The presence of heterobaric leaves in the higher strata of a forest can be advantageous because sclerenchymatous BSEs can give additional mechanical support, due to the strength given by the sclerenchyma cells (Dickson 2000;Cutler et al. 2008), and help to maintain leaf shape and volume (Roth 1984), as well as protect against herbivores (Sack & Scoffoni 2013).Secondarily, BSEs can perform optic functions such as facilitating the dispersion of light within the compartments of the leaf (Karabourniotis et al. 2000;Nikolopoulos et al. 2002), thereby enhancing photosynthetic rate (Nikolopoulos et al. 2002;Liakoura et al. 2009;Buckley et al. 2011).
Plants restricted to lower strata, on the other hand, are subjected to low levels of heterogeneous light (Théry 2001;Kenzo et al. 2007;Valladares & Niinemets 2008).These conditions are beneficial to homobaric leaves with their welldeveloped spongy parenchyma (Fig. 1D), as observed in the studied species [spongy:palisade parenchyma ratio for MOF homobaric leaves (2.1 ± 0.9); for MOF heterobaric leaves (1.5 ± 0.6); for LLODF homobaric leaves (5.3 ± 3.2) and for LLODF heterobaric leaves (3.9 ± 1.7)].A thicker spongy parenchyma is advantageous for capturing diffused light because the irregular-shaped cells reflect light rays within the mesophyll, thereby facilitating more efficient absorption (Vogelmann et al. 1996).Homobaric leaves also increase the proportion of photosynthetic areas in the mesophyll (Terashima 1992), which may contribute to more efficient photosynthesis and water use (Pieruschka et al. 2006;Pieruschka et al. 2010;Lynch et al. 2012) Thus, under limited light conditions, species with homobaric leaves perform better than those with heterobaric leaves (Kenzo et al. 2007).
The distribution of species with homobaric and heterobaric leaves was weakly correlated with taxonomic group.Although Lauraceae is present in all strata of LLODF and in the first three strata of MOF, it is the only family that is represented by a larger number of heterobaric leaf species.All the species of the families Aquifoliaceae and Melastomataceae, which are commonly found in under-canopy strata, had homobaric leaves.Even though they comprise species with both leaf types, the families Euphorbiaceae, Myrtaceae, Primulaceae, and Sapindaceae did not show a distributional pattern related to strata.The one exception was Fabaceae, whose species with heterobaric leaves were present in Stratum 3 in MOF.These results indicate that the leaf types of each species are more dependent on habitat and/or life form type than phylogenetic relationships.Environmental filters have convergent effects and seem to favor functional diversity due the habitat heterogeneity, especially in tropical forest communities (Manel et al. 2014).
In conclusion, the occurrence of homobaric and heterobaric leaves seems to be related to light stratification.The distribution of homobaric and heterobaric leaves in the different forest strata shows that light stratification acts as an ecological filter on the composition of the vegetation.Heterobaric leaves tend to occur in hotter strata that are more exposed to light, while homobaric leaves are more frequent in the under-canopy and more humid strata.This difference indicates that both leaf types occupy different positions on the "leaf economic spectrum", based on the balance between the cost of investiment in structural tissues and the investiment in photosynthetic tissues for carbon fixation via photosynthesis (sense Wright et al. 2004).
Besides environmental influences, the occurrence of leaf types is weakly related to taxonomic group.Only Lauraceae included a large number of heterobaric species.These results show that these two leaf types (homobaric/heterobaric) are more dependent on habitat and/or life form than phylogenetic relationships.Environmental filters seem to shape functional diversity due to habitat heterogeneity, especially in tropical forest communities.

Figure 1 .Figure 2 .
Figure 1.Surface and cross sections of homobric and heterobaric leaves.A. Surface of heterobaric leaf of Ocotea porosa, showing the division of the lamina in small areas by bundle sheath extension (BSE).B. Cross section of heterobaric leaf of Ocotea porosa, showing BSE. C. Surface of homobaric leaf of Lonchocarphus muehlbergianus, showing a homogeneous lamina.D. Cross section of homobaric leaf of Tibouchina sellowiana.Bars: A and C = 2 mm; B = 30 µm; D = 50 µm

Table 3 .
Presence and absence of bundle sheath extension (BSE) on tree species from Mixed Ombrophilous Forest, by stratum.
(Hammer et al. 2001)f each forest type were compared using t-test.Both analyses were performed on PAST software(Hammer et al. 2001).The independent distribution analysis of heterobaric leaves among forests and strata employed the χ² test (P < 0.05).Since MOF Stratum 4 included only one species, Araucaria angustifolia (Bertol.)Kuntze, it was excluded from this test, which aimed to verify the distribution of heterobaric leaves among plant families.The independent distribution analysis was performed using the RCMDR package (2, 1-7) for R program (version 3.1.2,R Foundation for Statistical computing, Vienna Austria).

Table 4 .
Number and percentage of species with homobaric and heterobaric leaves by forest type and stratum.Legend: LLODF -Lowland Ombrophilous Dense Forest; MOF -Mixed Ombrophilous Forest.

Table 5 .
Average height, average values and respective standard deviations of leaf area and leaf thickness by stratum, leaf types and forest type.Legend: LLODF -Lowland Ombrophilous Dense Forest; MOF -Mixed Ombrophilous Forest.(*) Leaf area and leaf thickness averages with the exclusion of leaves > 100 cm2, only in LLODF.Different letters in the same column, within the forest type, represent statistical difference, Tukey test (p<0.05).