Leaf and inflorescence axis anatomy of Brazilian species of Rapateoideae ( Rapateaceae , Poales )

The anatomy of leaves and inflorescence axes of Spathanthus (2 spp.), Rapatea (2 spp.), Cephalostemon (1 sp.), and Duckea (1 sp.) (Rapateoideae, Rapateaceae) was studied to identify useful characters for taxonomy. The cross-section shape of inflorescence axis differentiates the genera, while the cross-section shape and structure of leaf midrib has a specific value. The following characteristics are exclusive of Spathanthus: silica cells randomly distributed in the leaf epidermis; plicate chlorenchyma in the leaf blade; presence of fiber bundles in the mesophyll and in the inflorescence axis parenchyma. Spathanthus is also distinguished by the number, type and distribution of vascular bundles in the inflorescence axis. The genus Rapatea is characterized by the presence of stomata and silica cells only on the abaxial epidermis of the leaves and chlorenchyma composed of arm cells in the leaf blade. Characteristics with diagnostic value for Cephalostemon riedelianus are: leaf epidermal cells with straight to slightly sinuous walls in frontal view, inflorescence axes presenting a defined cortex, fiber bundles facing the larger vascular bundles and a fistulous pith. The anatomical characteristics of the leaves and inflorescence axes thus proved to be of taxonomic value in generic and specific levels. They are also useful to differentiate Rapateoideae from other subfamilies of Rapateaceae.


INTRODUCTION
Rapateaceae comprise approximately 100 species in 16-17 genera (Stevenson et al. 1998, Givnish et al. 2004) and are a basal family in Poales along with Bromeliaceae and Typhaceae (Linder andRudall 2005, Givnish et al. 2010).The family has a restricted distribution in South America, mostly in the Guayana Shield, with the exception of Mascalocephalus dinklagei that occurs in western Africa (Maguire 1958, Stevenson et al. 1998).In Brazil, approximately 39 species and nine genera are found, growing primarily in the Amazon region (Monteiro 2014).They are perennial herbs with a short stem from which distichous or spirodistichous leaves and inflorescence axes (scapes) originate (Maguire 1958, Stevenson et al. 1998).
Rapateaceae are divided into three subfamilies: Rapateoideae, Monotremoideae, and Saxofridericioideae (Givnish et al. 2004).Rapateoideae are the earliest divergent ones and comprise the ÂNGELA L. DALTIN, ALINE ORIANI and VERA L. SCATENA genera Spathanthus, Rapatea, Cephalostemon, and Duckea (Givnish et al. 2004).Spathanthus is the sister group of Rapatea and Cephalostemon (Givnish et al. 2004).These three genera were the first to diverge and are remarkable for presenting dispersion ranges that extend out of the Guayana Shield to the Amazon Basin (Givnish et al. 2000).
The genus Duckea (made up of four species) was described by Maguire (1958) and later synonymized as Cephalostemon by Steyermark (1988) due to their morphological similarities.However, Berry (2004), in his survey on Rapateaceae of the Venezuelan Guayana Flora, once again separates the genera based on the morphology of seeds, leaves and inflorescence axes.In the most recent phylogenetic study of the family (Givnish et al. 2004), which presents suprageneric taxonomic changes, Duckea is also treated as a separate genus.Duckea squarrosa, that was studied here, is one of three species of the genus that occurs in Brazil and it is endemic (Monteiro 2014).
Cephalostemon is made up of eight species, five of which are found in Brazil, and Cephalostemon riedelianus, that was studied here, is also endemic (Maguire 1958, Monteiro 2014).Spathanthus comprises two species, both occurring in Brazil (Maguire 1958, Monteiro 2014) and studied in this work.Rapatea comprises about 20 species (Stevenson et al. 1998), among which 14 occur in Brazil (Monteiro 2014), and two of them were also studied.
According to Carlquist (1969) who analyzed the anatomy of vegetative organs and inflorescence axis of 15 of the 17 genera of the family, the leaves in Rapateaceae are anatomically characterized by: paracytic stomata only on the abaxial surface; presence of silica cells in the epidermis; mesophyll differentiated into palisade and spongy tissue, the last composed of arm parenchyma; and slimeproducing trichomes in the leaf axils.
The inflorescence axes in Rapateaceae are characterized by: hypodermal fibers as a continuous sheath or as strands; a ground parenchyma composed of sphaeroidal or arm cells; presence of phenolic idioblasts; and vascular bundles nearer to the centre enclosed by a common fibrous sheath (Carlquist 1969).
Given the importance of identifying anatomical characteristics with taxonomic value, this paper aimed to describe the anatomy of leaves and inflorescence axes of Spathanthus bicolor, S. unilateralis, Rapatea paludosa, R. ulei, Cephalostemon riedelianus, and Duckea squarrosa (Rapateoideae) to characterize the subfamily and the genera, contributing to the understanding of infrafamiliar relationships.

MATERIALS AND METHODS
The anatomy of leaves and inflorescence axes was analyzed in six Brazilian species of Rapateoideae.The species studied, collecting areas and voucher numbers are presented in Table I.The vouchers were deposited in the Herbarium Rioclarense (HRCB) at Universidade Estadual Paulista -UNESP.The collected material was fixed in 50% FAA (37% formaldehyde, glacial acetic acid, 50% ethanol, 1:1:18 v/v) and subsequently stored in 70% ethanol (Johansen 1940) for the anatomical studies.Only for Duckea squarrosa the anatomical study was performed on herborized material, which was previously boiled in water and glycerin for tissue rehydration.We analyzed at least three individuals of each species, collected from different populations.
Cross, longitudinal, and paradermal sections in the median region of the leaves (blade and sheath) and inflorescence axes (scapes) were made by hand, using disposable razor blades.The ANATOMY OF RAPATEOIDEAE sections were double stained with basic fuchsin and astra blue (Roeser 1972) and mounted on semi-permanent slides with glycerin gelatin.The presence of phenolic compounds was tested with aqueous FeCl 3 (Johansen 1940).Cross and longitudinal sections were also obtained by using a microtome (Leica, RM 2245).For this purpose, leaf and inflorescence axis samples were dehydrated in an n-butyl alcohol series and embedded according to Feder and O'Brien (1968) in (2-hydroxyethyl)methacrylate (Leica Historesin Embedding Kit).These sections were stained with periodic acid, Schiff's reagent (PAS), and toluidine blue (O'Brien et al. 1964, Feder andO'Brien 1968) and mounted on permanent slides using Entellan (Merck).
The results were documented with a camera (Leica, DFC 450) coupled to a microscope (Leica, DM 4000B), using the LAS software (Leica Application Suite V.4.0.0).

LEAF
The variable characteristics of the studied species are presented in Table II.In frontal view, the epidermal cells have sinuous  or straight to slightly sinuous walls (Fig. 4).The stomata are tetracytic (Figs.3-4) and may occur on both sides or only on the abaxial leaf surface.When present on both sides, the stomata are more abundant on the abaxial surface.
All species have epidermal cells containing silica bodies that may be present on one or both leaf surfaces.In frontal view, these silica cells have a random distribution (Figs. 1 and 2 -arrows) or are distributed in longitudinal rows .

INFLORESCENCE AXIS
The anatomical characteristics that differentiate genera or species are presented in Table III.The cross-section shape of the inflorescence axis varies among the genera.It is triangular in Spathanthus (Figs. 35 and 38), elliptical in Rapatea (Figs. 42 and  47), and circular in Cephalostemon riedelianus (Fig. 52) and Duckea squarrosa (Fig. 55).In Rapatea, the inflorescence axis has two wings, one opposite to the other, which are of different sizes in R. paludosa (Figs.42 and 43) and of the same size with vascular bundles in R. ulei (Fig. 47).In C. riedelianus, the inflorescence axis presents ribs at the region of the larger vascular bundles (Figs.52 and 53).
All species present collateral vascular bundles surrounded by a double sheath: the outer sheath is composed of thin-walled cells, and the inner sheath is composed of thick-walled cells (Figs. 36,39,44,49,53 and 56).In Spathanthus, the vascular bundles are simple (Figs. 36 and 39), while in the remaining species there are simple or compound vascular bundles (Figs. 42,49,53 and 56).
Larger vascular bundles, in number of 5 to 10, have a circular distribution in Rapatea (Figs. 42  and 47) and in Duckea squarrosa (Fig. 55), while smaller vascular bundles are randomly distributed in the parenchyma.In Spathanthus (Figs. 35 and 38), the larger vascular bundles are more numerous (>10) and both, smaller and larger ones, are randomly distributed.In Cephalostemon riedelianus, the larger vascular bundles, in number of 5 to 10, alternate with the smaller ones, forming the ring that delimits a parenchymatous, fistulous pith (Fig. 52).

DISCUSSION
According to the results, the leaves of the studied species can be characterized by the presence of a single-layered epidermis with silica cells; tetracytic stomata; a regular, plicate, or arm chlorenchyma in the mesophyll; and collateral, double-sheathed vascular bundles.The leaf sheath has mucilagesecreting trichomes on the adaxial surface; a chlorenchyma composed of arm cells; and margin formed by the extension of the epidermis.
The inflorescence axes are characterized by having a single-layered epidermis with silica cells; tetracytic stomata; a parenchyma composed of isodiametric or arm cells delimiting air canals; phenolic idioblasts; and collateral vascular bundles.These features were also observed in the leaves and inflorescence axes of other representatives of Rapateaceae (Carlquist 1966, 1969, Ferrari et al. 2014) and may be used to describe the family.ÂNGELA L. DALTIN, ALINE ORIANI and VERA L. SCATENA According to Carlquist (1969), the presence of paracytic stomata only on the abaxial surface of the leaves is a distinctive feature of Rapateaceae.The results obtained here, however, showed that this characteristic varies among the genera, which may include species with stomata on both leaf surfaces.Our results also showed that the stomata are tetracytic, in agreement with the results obtained by Ferrari et al. (2014) for species of Monotrema, Stegolepis and Saxofridericia, therefore, this is probably a characteristic of the family.Carlquist (1969) also reported the presence of central vascular bundles enclosed by a common fibrous sheath in the inflorescence axes as a characteristic of the family.Never theless, in  most of the species studied here the cortex of the inflorescence axis is not defined and the vascular bundles are randomly distributed in the parenchyma.The presence of silica bodies, a common characteristic of leaves and inflorescence axes of all studied species, was also observed in other organs of Rapateaceae, such as stems, bracts and flowers (Carlquist 1966, 1969, Stevenson et al. 1998, Oriani and Scatena 2012).Carlquist (1966) used the presence of silica cells associated with subepidermal fiber strands in the stem to characterize the subfamily Rapateoideae.For the leaves and inflorescence axes, however, epidermal cells with silica bodies also occur in species that belong to different subfamilies (Carlquist 1969, Ferrari et al. 2014).The likely function of silica bodies is to reduce the transpiration and to improve photosynthesis, through efficient light distribution in the parenchyma (Prychid et al. 2004).They may also help maintain the rigidity of stems and inflorescence axes and reduce herbivory on leaves (Prychid et al. 2004).Silica bodies are present in representatives of other families of Poales as Poaceae, Cyperaceae, Thurniaceae, Bromeliaceae, Centrolepidaceae, Ecdeiocoleaceae, and Joinvilleaceae (Tomlinson 1969, Dahlgren et al. 1985, Silva and Alquini 2003, Prychid et al. 2004, Arruda and Neves 2005, Proença and Sajo 2007).They also occur in other commelinid families (included in Arecales, Zingiberales and Commelinales) and may represent a synapomorphy of the clade with several secondary reversals (Prychid et al. 2004).
The mucilage-secreting trichomes in the leaf sheath were reported for different genera of Rapateaceae (Carlquist 1969, Ferrari et al. 2014) and may represent an apomorphy of the family.The function of the mucilage may be to protect the leaves from solar irradiation and this feature may be related to the environment where the family arose, which was at the lowlands of the Guayana Shield (Givnish et al. 2000(Givnish et al. , 2004)).The mucilage production in leaves may even be related to different functions depending on the type of substance produced.It may protect the leaves not only from intense light, but also from herbivores and pathogens, extreme temperatures, and excessive water loss (Werker 2000).
Our results show that the following characteristics are exclusive of Rapateoideae: leaves and inflorescence axes with epidermal cells with slightly thickened outer periclinal walls and subepidermal fiber strands; vascular bundles of the leaf midrib region with a conspicuous fiber sheath on the phloem; and inflorescence axes with phenolic idioblasts in the epidermis and parenchyma.These features are not present in species of Monotrema (Ferrari et al. 2014), a genus previously included in the Rapateoideae (Maguire 1958), corroborating the actual circumscription of the subfamily (Givnish et al. 2004).
The data obtained in this study also show the occurrence of characteristics that delimit the genera.Spathanthus species have leaves with randomly distributed silica cells in the epidermis, fiber bundles scattered in the mesophyll, and a plicated chlorenchyma.In these species, the inflorescence axes are triangular in cross-section and present fibers bundles randomly distributed in the parenchyma and above ten simple vascular bundles as diagnostic characteristics.Rapatea species have leaves with stomata and silica bodies only on the abaxial epidermis and with a chlorenchyma composed of arm cells, besides elliptical and winged inflorescence axes.Cephalostemon is distinguished by presenting leaves with epidermal cells with straight to slightly sinuous walls in frontal view and ribbed inflorescence axes with a defined cortex, fiber bundles facing the larger vascular bundles, and a fistulous pith.
When we compare the studied genera, it may be noticed that Cephalostemon and Duckea share the greatest number of features, followed by Rapatea and Duckea.The anatomical similarities between Cephalostemon and Duckea reflect their morphological similarities, already reported by other authors who even synonymized Duckea as Cephalostemon (Steyermark 1988).The high similarity between Cephalostemon, Duckea, and Rapatea also corroborates the phylogenetic analysis of the family in which Cephalostemon and Rapatea appear as sister groups (Givnish et al. 2000(Givnish et al. , 2004)).
Besides delimiting the genera, some characteristics proved to be diagnostic for species such as the cross-section shape and structure of the leaf midrib.The size and composition of the wings present in the inflorescence axis of Rapatea differentiate the species from each other.Rapatea paludosa is also distinguished by presenting vascular bundle sheath extensions facing both leaf surfaces.ÂNGELA L. DALTIN, ALINE ORIANI and VERA L. SCATENA It may be emphasized that the anatomical characteristics identified in the leaves and inflorescence axes of Rapateoideae not only have taxonomic value but may also have an adaptive value.The presence of silica bodies in both organs and mucilage-producing trichomes in the leaf sheath are examples, whose probable function has been discussed previously.The presence of air canals in the leaves and inflorescence axes may also be related to environment.Since the species grow predominantly in wet or flooded areas (Berry 2004, Givnish et al. 2000), the presence of air canals may increase the aeration of these organs, thus facilitating gas exchange (Kawase and Whitmoyer 1980).
Arm cells delimiting air canals and fiber bundles scattered in the mesophyll are also present in the leaves of some species of Bromeliaceae (Proença andSajo 2007, Tomlinson 1969), corroborating the phylogenetic proximity between these two families, which have a basal position in Poales (Linder andRudall 2005, Givnish et al. 2010).