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MICROMORPHOLOGY AND ANATOMY OF LEAVES OF SYZYGIUM FLORIBUNDUM (MYRTACEAE: SYZYGIEAE), A RAINFOREST TREE ENDEMIC TO EASTERN AUSTRALIA
RETAMALES, H. A.1, SCHERSON, R.2, SCHARASCHKIN, T.1
Although species of Syzygium are abundant components of the rainforests in Queensland and New South Wales, little is known about the anatomy of the Australian taxa. Here we describe the foliar anatomy and micromorphology of Syzygium fl oribundum (syn: Waterhousea fl oribunda) using stan- dard protocols for scanning electron microscopy (SEM) and light microscopy. Syzygium fl oribun- dum possesses dorsiventral leaves with cyclo-staurocytic stomata, single epidermis, internal phloem, rhombus-shaped calcium oxalate crystals and complex-open midrib. In general, leaf anatomical and micromorphological characters are common with some species of the tribe Syzygieae. However, this particular combination of leaf characters has not been reported in a species of the genus. The anatomy of the species is typical of mesophytic taxa.
1 School of Earth, Environmental and Biological Sciences, Science and Engineering Faculty. Queensland University of Technology. Brisbane, QLD 4001, Australia. 2 Plant Biology Laboratory, Faculty of Forest Sciences and Nature Conservation, University of Chile, P.O. Box 9206, Santiago, Chile.
INTRODUCTION Craven & Biffi n, comb. nov. is now considered a Syzygium Gaertn. is a large genus in the family section in subgenus Acmena (DC.) Craven & Biffi n, Myrtaceae that comprises approximately 1200 species comb. nov., in the genus Syzygium. (Craven and Biffi n, 2010). Species of the genus are predominantly trees occurring in rainforests from Anatomical and micromorphological characters have China and southeast Asia to south eastern Australia shown to be relevant for taxonomic delimitation in (Hyland, 1983; Craven et al., 2006). These species are the family (Schmid, 1980; Fontenelle et al., 1994), abundant components in the upper and medium strata but only a few species have been examined. The of rainforests of eastern Australia (Hyland, 1983). leaf anatomy of a number of species of Syzygium, Syzygium fl oribundum F.Muell (sect. Waterhousea) but not S. fl oribundum, was investigated by Soh (Weeping Lilly Pilly) is a tree up to 30 m tall & Parnell (2011) and shown to be taxonomically (FIG. 1 A). It is endemic to Australia and occurs in useful for the infrageneric classifi cation of the southeastern Queensland and northeastern New South genus. The leaf surface micromorphology has not Wales, mainly along creeks and rivers in microphyll been described for any species in the genus. This rainforests (Hyland, 1983). The species is regarded as investigation aims to undertake a complete anatomical a horticulturally important tree, adequate for streets and micromorphological description of the leaves of and gardens and appears to be relatively tolerant to Syzygium fl oribundum so as to determine if there myrtle rust (Queensland Government, 2014). As with are any taxonomically important characters that other species of Myrtaceae, it is also important for differentiate this species from others in the genus. local fauna as a food source, particularly birds and insects (Wilson, 2011). MATERIAL AND METHODS SAMPLING Syzygium fl oribundum was formerly placed in the Mature leaves of Syzygium fl oribundum were collected genus Waterhousea B. Hyland, which was recognized as fresh material from the natural habitat of the as an exclusively Australian genus with four species. species (FIG 1 B). Leaves were collected from sun- It has recently been transferred to Syzygium on the exposed branches from randomly selected individual basis of molecular and morphological phylogenetic trees in a riverside forest in Enoggera, QLD (27° 26’ evidence (Biffi n et al., 2006; Craven et al., 2006; 27” S / 153° 0’ 45” E) and a small rainforest 2 km Craven & Biffi n 2010). Waterhousea (B.Hyland) away from the coast in Noosa, QLD (26° 38’ 52” S / 46 PROCEEDINGS OF THE ROYAL SOCIETY OF QUEENSLAND
153° 2’ 36” E). Specimens (Reta-056.1, Reta-056.2, LIGHT MICROSCOPY Reta-056.3 and Reta-056.4) are currently housed at Leaves were fi xed in FAA for 24-48h prior to being the Queensland Herbarium (BRI). The location of dehydrated through a graded ethanol series and sampling localities and the geographical distribution embedded in paraffi n wax (Johansen, 1940; Ruzin, of the species are shown in the FIG 2. 1999). Transverse sections (5μm thickness) were cut using a Leica rotary microtome. Staining of sections SCANNING ELECTRON MICROSCOPY was performed using a 0.05% (w/v) of ruthenium red Leaves were fi xed in FAA for 24-48h, dehydrated in distilled water and a counterstaining with 0.1% using a graded ethanol series and then critical point (w/v) solution of toluidine blue (TBO) in distilled dried (Anderson, 1951) in an Autosamdri-815 water (O’Brien et al., 1964; Chaffey et al., 2002). automatic critical point drier. Samples were mounted Leaf clearings were prepared by immersing tissue on stubs with self-adhesive double-sided carbon discs fragments of 1-2 cm2 in 10% KOH at room temperature and sputter-coated with gold palladium for 175 sec for 48 h followed by 7% NaClO for 2 h or until leaves using a Leica EM SCD005 Gold Coater. Examination turned transparent. Cleared leaves were washed fi ve and photography were conducted using a FEI Quanta times with distilled water, stained with 0.1% safranin 200 SEM/ESEM operated at 10kV. and mounted with lactoglycerol (lactic acid-glycerol
FIG. 1. Syzygium fl oribundum, habit and leaf morphology. A, Tree. B, Detail of leaves. FIG 2. Geographical distribution of S. fl oribundum showing sampling localities. Red dots- geographical distribution of the species. Green dots- sampling localities. Map obtained and adapted from: Australia’s Virtual Herbarium (2014). MICROMORPHOLOGY AND ANATOMY OF LEAVES OF SYZYGIUM FLORIBUNDUM 47 (MYRTACEAE: SYZYGIEAE), A RAINFOREST TREE ENDEMIC TO EASTERN AUSTRALIA
1:1). Sections were dried and mounted with Entellan RESULTS mounting medium. Slides were observed using a GROSS MORPHOLOGY OF LEAVES Nikon SMZ 800 Stereo light microscope (Nikon The leaves of S. fl oribundum are simple, opposite, eclipse 50i compound) and images captured using the lanceolate-elliptical in shape. The apex is acuminate NIS Elemental digital image analysis software. and the base attenuate. Both surfaces are punctuated. Leaves have numerous secondary and tertiary reticulate TERMINOLOGY veins. Venation is pinnate and weakly to strongly Terminology for general anatomical descriptions, brochidodromus. Intramarginal veins are visible on including glands, stomatal complex types and cell both adaxial and abaxial surfaces (FIG. 3 A). types were based on Esau (1953) and Gifford and Foster (1989). Myrtaceae-specifi c terminology was CUTICLE, EPIDERMAL CELLS AND STOMATA based on previous work on this family (Schmid, 1980, The adaxial cuticle is thicker than the abaxial cuticle. 1984; Keating, 1984; Cardoso et al., 2009; Soh & The cuticle contains polyphenols as indicated Parnell, 2011 and da Silva et al., 2012). by bluish-green staining with TBO. Epicuticular
FIG. 3. Light (LM) and scanning electron micrographs (SEM) of leaves of S. fl oribundum. A, Leaf clearing showing reticulate tertiary venation. B, SEM micrograph showing distribution of stomata on the leaf surface. C, SEM micrograph showing detail of cyclo-staurocytic stomata and surrounding epidermal cells. D, SEM micrograph of secretory cavity on the abaxial surface showing overlying cell (arrow) surrounded by 6-8 elongated cells. Scale bars = 500 μm in A, 20 μm in C. 48 PROCEEDINGS OF THE ROYAL SOCIETY OF QUEENSLAND waxes are mainly granules and fl akes and randomly leaf, are abundant throughout the mesophyll and have distributed on the surface (FIG. 3 B). variable dimensions (62 ± 25 mm of diameter). They are composed of large spaces surrounded by a sheath Adaxial and abaxial epidermal cells are small, of peripheral epithelial cells. These cells are almost compressed, plano-convex and mainly isodiametric. completely disintegrated in mature leaves (FIG. 4 E). Adaxial epidermal cells are rounded and have In paradermal view, secretory cavities are observed as straight cell walls, while abaxial epidermal cells are one overlying cell surrounded for 6-8 elongated cells. irregularly rounded and the anticlinal cell walls are There are no stomata in the vicinity of the secretory strongly sinuous (FIG. 3 C). cavities (FIG. 3 D).
Stomatal complexes are cyclo-staurocytic and DISCUSSION randomly distributed on the abaxial surface of the Syzygium fl oribundum shares a number of anatomical leaf (Fig. 3 B), but also can occur in groups above and micromorphological characters with other midrib. Guard cells are kidney-shaped and the pore Myrtaceae. Some of these features include the between them is narrow at the equatorial region. presence of calcium oxalate crystals, internal phloem Stomata are circular to elliptical and are located at and schizogenous secretory cavities. Anatomical the same level of the epidermal cells. The length of characters described here largely agree with Keating the guard cells is 8–13 mm long. (1984) and Soh & Parnell (2011) for Syzygium/ Waterhousea. Nonetheless, the combination of some MIDRIB features such as cyclo-staurocytic stomata, rhombus- The midrib of S. fl oribundum is a mixture of closed shaped crystals, midrib with a well-developed adaxial and complex open, with two or more vascular bundles phloem partition and poorly developed incurved packed in the centre of the leaf (FIG. 4 A, B). The margins of the phloem are not found in other species leaf blade is impressed on the adaxial surface above of the genus. the midrib. The bundle sheath extension is composed of rounded-polygonal cells and is clearly developed, Calcium oxalate crystals have been reported in particularly on the abaxial side (FIG. 4 A). The midrib practically all the Myrtaceae studied (Cardoso et is arc-shaped with a deep curvature. The abaxial al., 2009; Gomes et al., 2009; Soh & Parnell, 2011). side is convex and the adaxial is slightly concave to However, rhombus-shaped calcium oxalate crystals as straight (FIG. 4 B). Fibres are continuous around the those found in this study have been reported in a few midrib. Xylem vessels of the midrib show scalariform Myrtaceae, mainly some species of Syzygium (Soh & perforation plates and helical wall thickenings. This Parnell, 2011). The function of these crystals is not species has internal phloem, with a well-developed completely clear, but has been related to the regulation adaxial phloem partition. Incurved margins of the of calcium and other minerals (Volk et al., 2002), as phloem are poorly developed. Phloem sieve tubes and well as protection against herbivores and pathogens companion cells have thin primary cell walls. Phloem (Franceschi & Nakata, 2005; Korth et al., 2006). fi bres have evident and thick secondary cell walls. Internal phloem was found in all vascular bundles MESOPHYLL of leaves, either as continuous tissue or as different The mesophyll is dorsiventral and is formed by degrees of adaxial phloem partition. This feature a 1-2 layered palisade parenchyma and a spongy is regarded as a typical anatomical character in parenchyma with abundant intercellular spaces. The the order Myrtales (Takhtajan, 1980; Cronquist. palisade parenchyma layer is somewhat loose and 1981) and is widely present in Myrtaceae (Schmid, composed of rectangular, attenuated and vertical 1980; Cardoso et al., 2009). Further investigation is cells. These cells possess thin primary cell walls needed to determine whether the internal phloem is and numerous chloroplasts. The spongy parenchyma derived from the procambium, or from mesophyll is composed of irregular shaped cells (rounded to cells (ground tissue). This developmental difference star-shaped). Both palisade and spongy parenchyma is regarded as a potentially informative taxonomic contain mucilage (FIG 4 C). Subepidermal idioblasts character (Patil et al., 2009). with rhombus-shaped calcium oxalate crystals occur throughout the palisade parenchyma (FIG. 4 D). Secretory cavities follow the typical schizogenous Secretory cavities are present on both sides of the pattern commonly observed in Myrtaceae (Alves et MICROMORPHOLOGY AND ANATOMY OF LEAVES OF SYZYGIUM FLORIBUNDUM 49 (MYRTACEAE: SYZYGIEAE), A RAINFOREST TREE ENDEMIC TO EASTERN AUSTRALIA
FIG. 4. Transverse light micrographs (LM) of leaves of S. fl oribundum. A, General view of the anatomy of the leaf blade showing slight depression above the midrib. B, Detail of the midrib showing xylem (stained green-blue), phloem (pink) and fi bres (purple). C, Dorsiventral mesophyll showing palisade and spongy parenchyma. D, Detail of the mesophyll containing subepidermal idioblasts with rhombus-shaped calcium oxalate crystals. E, Secretory cavity composed of a large space surrounded by epithelial cells. cr- calcium oxalate crystals, fi - fi bres, ph- phloem, pp- palisade parenchyma, sc- secretory cavities, sp- spongy parenchyma, xy- xylem. Scale bars = 100 μm 50 PROCEEDINGS OF THE ROYAL SOCIETY OF QUEENSLAND al., 2008; Donato & Morretes, 2009; Gomes et al., help and technical advice. Thanks to members of the 2009). The schizogenous pattern can be identifi ed by Plant Structure and Systematics group at QUT for the presence of elongated epithelial cells surrounding feedback and advice. Funding for this investigation the secretory space in mature leaves (Cicarelli et has been provided for CONICYT-Government of al. 2008). Other patterns identifi ed in Myrtaceae Chile and QUT. include schizolysigenous secretory cavities (a mixture of schizogenous and lysigenous, the latter due to LITERATURE CITED disintegration of cells) in Myrtus communis (Cicarelli ALVES, E.; TRESMONDI, F. & LONGUI, E. 2008. et al. 2008). Secretory cavities are the structures Análise estrutural de folhas de Eugenia unifl ora referred by Hyland (1983) as oil dots on the surface L. (Myrtaceae) coletadas em ambientes rural e of the leaves, which are regarded as very numerous. urbano, SP, Brasil. Acta Botanica Brasilica 22 (1): Volatile oils secreted by these structures in Myrtaceae, 241-248. have been identifi ed as fl avonoids (Wollenweber et al., ANDERSON, T. 1951. Techniques for the 2000) and terpenoids (Tanaka et al., 1996; Lee, 1998; preservation of three-dimensional structure in Judd et al., 1999). Chemical compounds produced by preparing specimens for the electron microscope. secretory cavities were not characterized in this study. Transactions of the New York Academy of Sciences 13: 130. Leaf anatomical characters are generally regarded as AUSTRALIA’S VIRTUAL HERBARIUM. 2014. homoplaseous in the genus Syzygium. However some Map output. Council of heads of Australasian have been shown to be useful for species identifi cation, Herbaria. Viewed 02 of September 2014.
characterization of the foliar colleters in Polychromatic staining of cell walls by Toluidine Myrtoideae (Myrtaceae). Australian Journal blue O. Protoplasma 59: 368-373. of Botany 60: 707-717. PATIL, V. S.; RAO, K. & RAJPUT, K. 2009. DONATO, A. & MORRETES, B. 2009. Anatomía Development of intraxylary phloem and foliar de Eugenia fl orida DC. (Myrtaceae). internal cambium in Ipomoea hederifolia Revista Brasileira de Farmacognosia 19: 759-770. (Convolvulaceae). Journal of the Torrey Botanical ESAU, K. 1953. Plant anatomy. (John Wiley & Sons, Society 136: 423-432. Inc, New York). QUEENSLAND GOVERNMENT, 2014. Business FONTENELLE, G.; COSTA, G. & MACHADO, and Industry Portal – Known plants affected by R. 1994. Foliar anatomy and micromorphology myrtle rust. URL:
AUTHOR PROFILES Hernan Retamales is PhD candidate at QUT working on the systematics of Australasian and South American Myrtaceae using molecular and morpho-anatomical data. His PhD is funded by the Government of Chile (CONICTY). Dr Rosa Scherson is a Lecturer at the School of Forestry in the University of Chile and is the external supervisor of Mr Retamales. Dr Tanya Scharaschkin is a Senior Lecturer at the School of Earth, Environmental and Biological Sciences at QUT. She has recused herself from the review and editorial process for this article, due to confl ict of interest as Editor of PRSQ, which has been handled by Dr. Geoff Edwards (President RSQ) instead.