Leaf Anatomy and Its Contribution to the Systematics of Aechmea Subgenus Macrochordion (de Vriese) Baker (bromeliaceae)

Leaf anatomy and its contribution to the systematics of Aechmea subgenus Macrochordion (de Vriese) ABSTRACT The leaf anatomy of the species Aechmea subgenus Macrochordion was analyzed to obtain valuable data on their taxonomic delimitation and to identify anatomical adaptations to their respective habitats and habits. All leaves of these species are hypostomatic, and present: peltate trichomes on both surfaces; stomata sunk in epidermal depressions; small epidermal cells with thick walls and inclusions of silica bodies; a mechanical hypodermis; an aquiferous parenchyma; chlorenchyma with fibrous clusters and air channels; and vascular bundles surrounded by a parenchymatic sheath and a cap of fibers. The results are evaluated within an adaptive and taxonomic context. Variations in hypodermic thickening, amount of water parenchyma, position of the air channels and shape of the cells filling the air channels are useful for delimiting groups of species, strengthening the relationships suggested by their external morphology.


INTRODUCTION
The Bromeliaceae includes 3,172 species (Luther 2008) and is a representative monocot family for Neotropical flora.It occupies a basal position within the Poales order and is closely related to Typhaceae and Rapateaceae (APG III 2009).Bromeliads demonstrate remarkable ecological versatility, inhabiting mesic to xeric environments (e.g.humid tropical forests to deserts) and display a wide range of terrestrial, lithophytic and epiphytic habits (Smith 1934, Benzing 2000a).Some morphological and physiological adaptations to drier habitats and particular life forms out of the soil include the presence of a central tank (which allows the plant to collect water and organic material inside enlarged and overlapping leaf sheaths), leaves with absorbing scales (the most distinguishing feature of the family, also referred to as peltate trichomes), water storage and mechanical support tissues, the development of 'crassulacean acid metabolism' (CAM) photosynthesis, and the progressive structural and functional reduction of root systems in epiphytic and rupiculous species (Tomlinson 1969, Benzing 1976, 2000a, b).
In Aechmea, potentially useful diagnostic data are still limited and most characters traditionally emphasized in previous taxonomic treatments of the genus (e.g.inflorescence, sepal and floral bract features, pollen grains morphology and petal appendages presence) have often failed to delimit natural groups (Faria et al. 2004, Schulte andZizka 2008).Thus, an investigation of different characters besides the ordinary external morphology (e.g.anatomical aspects) became essential to provide additional and valuable data to increase knowledge about the systematics of the genus.The function and adaptive value of anatomical features are often extremely useful as they may help to reveal more clearly the homologies of structures for classification purposes and reconstruction of phylogeny (Stuessy 1990).Structural aspects of some vegetative organs, for example, have contributed to the generic/infrageneric delimitation of many groups (Weiner and Liese 1993, Starr and Ford 2001, Alves et al. 2002, Calvente et al. 2008, Oliveira et al. 2008, Cardoso et al. 2009).
The taxonomic and phylogenetic use of anatomical characters, especially leaves´, has been helpful in the delimitation of different genera of Bromeliaceae (Robinson 1969, Varadarajan and Gilmartin 1988, Gilmartin et al. 1989, Sajo et al. 1998, Almeida et al. 2009).For Aechmea, foliar anatomical aspects have been investigated in descriptive works (Tomlinson 1969, Smith and Downs 1974, Flores 1975, Braga 1977, Benzing 2000b), using an ecophysiological approach (Scarano et al. 2002), or used as a tool for taxonomic delimitations of the subgenera Lamprococcus (Aoyama and Sajo 2003) and Chevaliera (Sousa et al. 2005) and for species of the genus occurring in São Paulo state, southeast Brazil (Proença and Sajo 2004).
Aechmea subgenus Macrochordion, the focus of this study, is characterized by grouping epiphytes, terrestrials and rupicolous medium-sized species, presenting simple, densely floccose or lanate inflorescences with sessile and polystichous flowers; entire carinate and unarmed floral bracts; unarmed and connate sepals, and well-developed fringed petal appendages (Faria et al. 2010).The species share great morphological similarity, which makes delimitation difficult, especially when some diagnostic characters are poorly preserved or not mentioned on the labels of herbarium specimens (e.g.calyx and corolla color).The subgenus is entirely restricted to the Brazilian Atlantic Forest, except for A. bromeliifolia, which occurs from Central America to Argentina.In a recent taxonomic revision, Faria et al. (2010)  The present study describes the foliar structure of Aechmea subgenus Macrochordion with the aim of providing additional morphological characters to support the systematic boundaries of the species, as well as identifying anatomical adaptations to their respective habitats.

MATERIALS AND METHODS
Leaves were obtained from cultivated specimens at the Universidade Federal do Rio de Janeiro (adult shoots from individuals collected in the field and maintained in a greenhouse).A voucher for each studied sample was deposited in the RFA herbarium under the following specifications: A. alba, Porto Seguro, Bahia, Faria et al. 139;A. bromeliifolia  Samples from the middle region of adult leaf blades were fixed in a solution of formaldehydeacetic acid-alcohol 50% and then transferred and stored in 50% alcohol (Johansen 1940).Transverse freehand sections were obtained using a razor blade, bleached with sodium hypochlorite and stained with Astra blue-Safranin (Bukatsch 1972).The histological slides were mounted in 50% glycerine and observed with an Olympus CH 30 light microscope.For histochemistry, fresh sections were submitted to the following tests: Lugol for starch (Johansen 1940), Sudan IV for lipids (Gerlach 1984) and acidic phloroglucin for lignin (Sass 1951).

RESULTS
The leaf epidermis of Aechmea subgenus Macrochordion species is organized in a single layer of cells and in transverse section can be almost plane on both surfaces, as in A. lamarchei (Fig. 1A), undulating on the abaxial surface in A. bromeliifolia var.albobracteata (Fig. 1B) and A. triangularis (Fig. 1C) or on both surfaces, as in A. bromeliifolia var.bromeliifolia (Fig. 1D), A. alba (Fig. 1E) and A. maasii (Fig. 1F).All epidermal cells have a reduced lumen with very thick and lignified anticlinal and internal periclinal walls (Fig. 2A), including one nearly spherical silica body (Fig. 2F).The external periclinal walls are covered with a thin cuticle, except in A. triangularis, which possesses a dense covering (Fig. 2G).All taxa have peltate trichomes on both surfaces, with the stalk composed of two cells sunk into epidermal depressions (Fig. 2A) and hypostomatic leaves with sunken stomata below the other ordinary epidermal cells (Fig. 1A-F, Fig. 2B).
All taxa have an adaxial and abaxial hypodermis.This tissue is more developed adaxially and executes a mechanical function due to cell wall thickening.The mechanical hypodermis is arranged in one or two layers of thickened or slightly thickened cells (Table I).Slightly thickened cells are observed in A. alba (Fig. 2C), A. maasii (Fig. 2D) and A. lamarchei (Fig. 2E) and thick-walled cells in A. bromeliifolia var.bromeliifolia (Fig. 2F), A. bromeliifolia var.albobracteata and A. triangularis (Fig. 2G).The cells are nearly isodiametric, except in A. triangularis, where the first layer of the adaxial surface presents anticlinally extended hypodermic cells (Fig. 2G).Lignified walls are also observed only in A. triangularis, and the remaining taxa exhibit a non-lignified hypodermis.
All taxa have a water parenchyma formed by cells without chloroplasts and with thin cellulosic walls, located below the adaxial mechanical hypodermis (Fig. 1A-F).The number of layers of water parenchyma varies from one to five (Table I), being more developed in A. bromeliifolia var.bromeliifolia, A. triangularis and A. bromeliifolia var.albobracteata (Fig. 1B-D).Below the water parenchyma, the chlorenchyma occupies the greater portion of the mesophyll (Fig. 1A-F) and is formed by anticlinally extended cells arranged like a palisade on the adaxial surface of A. bromeliifolia var.albobracteata (Fig. 1B, Table I) and on both surfaces of A. triangularis (Fig. 1C, Table I).In the remaining taxa, chlorenchymatic cells are slightly extended anticlinally or are almost isodiametric (Fig. 1A, D-F, Table I).Air channels (or air-lacunae in the sense of Tomlinson 1969) alternate with the vascular bundles are also observed in all taxa (Fig. 1A-F).In A. alba, A. lamarchei and A. maasii, the air channels are usually continuous to the substomatal chambers on the abaxial surface and filled with nearly isodiametric cells with short projections (Fig. 1A, E, F, Fig. 3B, Table I).In A. triangularis, A. bromeliifolia var.albobracteata and A. bromeliifolia var.bromeliifolia, the air channels are farther from the abaxial surface, with no direct connection to the substomatal chambers, and are filled with stellate cells (Figs. 1B-D, 3A, Table I).Fibrous clusters have a scattered distribution adaxially and abaxially in the chlorenchyma of all examined species (Fig. 1A-F).In all taxa we observed a single series of collateral vascular bundles arranged in the central portion of the mesophyll (Fig. 1A-F), along with slender commissural bundles (Fig. 3G).The larger vascular bundles are surrounded by slightly thickened and lignified cells (also between the xylem and phloem) and covered by a cap of fibers on the xylem and phloem poles (Fig. 3C, E).This cap of fibers can be totally lignified in A. alba, A. lamarchei and A. maasii or incompletely lignified in A. bromeliifolia var.bromeliifolia, A. bromeliifolia var.albobracteata and A. triangularis (Table I).The slightly thickened and lignified cells surrounding the smaller vascular bundles do not appear between the xylem and phloem and the cap of fibers can extend to both poles (Fig. 3D) or not (Fig. 3F).In association with the lignified cells and the cap of fibers, we observe a sheath of parenchymatic cells contained starch grains (Fig. 1A-F; 3C-F), smaller than the remaining chlorenchymatic cells of the mesophyll.

DISCUSSION
The anatomical foliar features of Aechmea subgenus Macrochordion are in accordance with the general patterns observed for Bromeliaceae (Tomlinson 1969, Smith andDowns 1974,

Benzing 2000b
).The parenchymatic sheath and fibers involving the vascular bundles are treated as the endodermis and the pericycle, respectively, following the nomenclature of Van Fleet (1961).P.B. Pita (unpublished data), Sajo et al. (1998), Arruda and Costa (2003) and Scatena and Segecin (2005) have also described these structures in other bromeliad species.Although Smith and Downs (1974) suggested that a palisade chlorenchyma is not a common character in the Bromeliaceae, Proença and Sajo (2004) reported the presence of this tissue in an exemplar of A. bromeliifolia from São Paulo state, southeastern Brazil.The authors, however, did not specify whether the specimen analyzed  (Faria et al. 2010), and in this study, we observed anticlinally extended cells arranged like a palisade in var.albobracteata.
The species analyzed show several xeromorphic characters commonly reported for Bromeliaceae (e.g.epidermal cells with lignified thick walls and silica body inclusions, mechanical hypodermis, water parenchyma, sunken stomata and lignified fibers in the mesophyll).Silica bodies have been associated with resistance against herbivory due to their inedibility (MacNaughton et al. 1985, Prychid et al. 2004), but are also important for refracting excess of light, helping individuals to establish in sun-exposed environments (Krauss 1949).These inclusions are also commonly found in other monocots families within Poales, suggesting that this is a plesiomorphic character for the order (Pereira et al. 2011).
According to Tomlinson (1969), sunken stomata below the remaining epidermal cells help to prevent dehydration by transpiration.Several plant families from arid zones, particularly succulents, have stomata deeply sunken in depressions in the epidermis (crypts), such as Agavaceae (Fahn and Cutler 1992) and Cactaceae (Calvente et al. 2008).However, this trait is not restricted among plants exposed to drought  (Faria et al. 2010).Previous studies also indicate the occurrence of sunken stomata in other Aechmea species from mesic environments (Proença andSajo 2004, Sousa et al. 2005) suggesting that the presence of this character may be not a significant adaptation to environmental conditions of low water availability.As discussed by and Sajo ( 2004), although many xeromorphic characters found in the Bromeliaceae allow the exploration of extreme environments, they can represent ancestral adaptations selected during the diversification of the family and should not be interpreted only as traits related to a particular environment where they currently live.
Besides the position below the other ordinary epidermal cells, the stomata of all Macrochordion taxa are restricted to the abaxial surface.Hypostomatism is a common condition for Bromeliaceae (Tomlinson 1969) and was also reported for other Aechmea species from subgenera Lamprococcus (Aoyama and Sajo 2003), Platyaechmea, Pothuava, Ortgiesia (Proença and Sajo 2004) and Chevaliera (Sousa et al. 2005).According to Fahn and Cutler (1992) stomata restricted to the abaxial surface are more common in plants from humid environments.Other authors have suggested that hypostomatic leaves may be a strategy to minimize water loss by convection currents or breezes that could remove water vapor from the leaf surface (Nobel 1999).Scatena and Segecin (2005) investigated the foliar anatomy of some Tillandsia (Tillandsioideae) in which xeromorphic traits are predominant, and argued that the presence of hypostomatic leaves in these species, may be more of a plesiomorphic condition retained than a response to environmental selection.
The hypodermis, along with the thick and lignified epidermal cells and the vascular and extravascular fiber bundles develop a mechanical function of strengthing the foliar structure.These tissues can also protect against hydric and temperature stress by reducing water evaporation from internal tissues and preventing mesophyll collapse in unfavorable abiotic conditions (Krauss 1949, Brighigna et al. 1984, Fahn and Cutler 1992).Besides avoiding water loss, the water parenchyma also protects the chlorenchymatic cells against excessive luminosity (Brighigna et al. 1984).The thick-walled hypodermic cells and greater amount of water parenchyma observed in A. bromeliifolia var.bromeliifolia, A. bromeliifolia var.albobracteata and A. triangularis may be adaptations to the particular environmental conditions and habits of these species.The main habitats occupied by A. bromeliifolia var.bromeliifolia and A. bromeliifolia var.albobracteata are dry and/or sun-exposed (Faria et al. 2010), such as the rocky grasslands (campos rupestres), caatingas and savannas (cerrados).Although A. triangularis is restricted to humid habitats of the Atlantic Forest (Faria et al. 2010), it grows only as epiphyte, and mostly in the higher strata of trees.Besides the water stressful condition created by the epiphytic life form, this species receives intense levels of luminosity.The remaining taxa (A. alba, A. maasii and A. lamarchei) grow as terrestrials or epiphytes, preferentially in humid and/or shaded habitats of dense ombrophile and semideciduous forests, shrubby and wooded sandy coastal plains (restingas) and tableland forests (Faria et al. 2010).Consequently, their foliar structures have a less well-developed water parenchyma and the cells of the mechanical hypodermis have thinner walls.Aechmea alba, A. lamarchei and A. maasii are also characterized by the air channels contiguous to the substomatal chambers, and the same character was reported for other Aechmea species from mesic habitats studied by Aoyama and Sajo (2003), Proença and Sajo (2004) and Sousa et al. (2005).Tomlinson (1969) argued that the air channels facilitate ventilation in species from mesic environments and are usually connected to the substomatal chambers LEAF ANATOMY OF Aechmea SUBGENUS Macrochordion in leaves of mesophytic bromeliads.In the subgenus Macrochordion, the occurrence of this character may also suggest an adaptation of the species to their respective habitats.
As observed by Versieux et al. ( 2010) for closely related species of the genus Alcantarea (Tillandsioideae), most of the anatomical features examined in this study are also very homogeneous within the Aechmea subgenus Macrochordion.However, some of the characters that were investigated are useful to characterize particular taxa, as well as circumscribing groups of species, strengthening the relationships indicated by their external morphology.Aechmea triangularis exhibits unique anatomical traits, such as a thick cuticle layer on the epidermal surface, anticlinally extended and sclerified hypodermic cells, and a palisade chlorenchyma on both faces of the leaf.Some aspects of the vegetative and reproductive external morphology also distinguish A. triangularis from other Macrochordion species, such as leaves with caudate and recurvate apices and a blue corolla (Faria et al. 2010).The leaf structure of A. triangularis is more similar to the same observed for both varieties of A. bromeliifolia due to the presence of a well-developed water parenchyma, hypodermic cells with thick walls, air channels not connected to the substomatal chambers and filled with stellate cells, and vascular bundles covered by a cap of incompletely lignified fibers.The close relationship among these taxa is also reflected by some aspects of their external morphology, such as the presence of leaf spines longer than 3 cm, emarginate, symmetric to slightly asymmetric and half connate sepals, and spatulate petals with emarginated apices (Faria et al. 2010).Similarly, A. alba, A. lamarchei and A. maasii have leaf spines up to 3 mm long, obtuse and distinctly asymmetric sepals and lingulate petals with obtuse apices (Faria et al. 2010).These species also share some anatomical features not observed in A. bromeliifolia and A. triangularis, such as a less developed water parenchyma, hypodermic cells with thinner walls, air channels filled with nearly isodiametric cells with short projections and connected to the substomatal chambers, and vascular bundles covered by a totally lignified cap of fibers.Variations in the shape of the cells interrupting the air channels were also reported by Aoyama and Sajo (2003) and Sousa et al. (2005), and showed to be useful in the delimitation of species within Aechmea subgenera Lamprococcus and Chevaliera.
This study contributed to a better understanding of some anatomical traits responsible for the adaptation of the Macrochordion species to their respective habitats and habits, as well as revealed some useful taxonomical characters to characterize particular species and to delimit groups of species.We also contributed with potential data to be explored in future phylogenetic studies within the subgenus and between Aechmea and other related Bromelioideae genera.