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Retamales, Hernan A., Scherson, Rosa, & Scharaschkin, Tanya (2014) Micromorphology and anatomy of leaves of Syzygium ﬂoribundum (Myr- taceae: Syzygieae), a rainforest tree endemic to eastern Australia. Proceedings of the Royal Society of Queensland, 119(1), pp. 45-52.

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Notice: Changes introduced as a result of publishing processes such as copy-editing and formatting may not be reﬂected in this document. For a deﬁnitive version of this work, please refer to the published source: 1 Micromorphology and anatomy of leaves of Syzygium floribundum (Myrtaceae:

2 Syzygieae), a rainforest tree endemic to eastern Australia

4 Hernan A. RetamalesA, Rosa SchersonB, Tanya ScharaschkinA

6 ASchool of Earth, Environmental and Biological Sciences, Science and Engineering Faculty.

7 Queensland University of Technology. Brisbane, QLD 4001, Australia.

8 BPlant Biology Laboratory, Faculty of Forest Sciences and Nature Conservation, University

9 of Chile, P.O. Box 9206, Santiago, Chile.

29 ABSTRACT

30 Although species of Syzygium are abundant components of the rainforests in Queensland and

31 New South Wales, little is known about the anatomy of the Australian taxa. Here we describe

32 the foliar anatomy and micromorphology of Syzygium floribundum (syn: Waterhousea

33 floribunda) using standard protocols for scanning electron microscopy (SEM) and light

34 microscopy. Syzygium floribundum possesses dorsiventral leaves with cyclo-staurocytic

35 stomata, single epidermis, internal phloem, rhombus-shaped calcium oxalate crystals and

36 complex-open midrib. In general, leaf anatomical and micromorphological characters are

37 common with some species of the tribe Syzygieae. However, this particular combination of

38 leaf characters has not been reported in a species of the genus. The anatomy of the species is

39 typical of mesophytic taxa.

41 INTRODUCTION

42 Syzygium Gaertn. is a large genus in the family Myrtaceae that comprises approximately

43 1200 species (Craven and Biffin, 2010). Species of the genus are predominantly trees

44 occurring in rainforests from China and southeast Asia to south eastern Australia (Hyland,

45 1983; Craven et al., 2006). These species are abundant components in the upper and medium

46 strata of rainforests of eastern Australia (Hyland, 1983). Syzygium floribundum F.Muell (sect.

47 Waterhousea) (Weeping Lilly Pilly) is a tree up to 30 m tall (FIG 1 A). It is endemic to

48 Australia and occurs in southeastern Queensland and northeastern New South Wales, mainly

49 along creeks and rivers in microphyll rainforests (Hyland, 1983). The species is regarded as a

50 horticulturally important tree, adequate for streets and gardens and appears to be relatively

51 tolerant to myrtle rust (Queensland Government, 2014). As with other species of Myrtaceae,

52 it is also important for local fauna as a food source, particularly birds and insects (Wilson,

53 2011).

55 Syzygium floribundum was formerly placed in the genus Waterhousea B. Hyland, which was

56 recognized as an exclusively Australian genus with four species. It has recently been

57 transferred to Syzygium on the basis of molecular and morphological phylogenetic evidence

58 (Biffin et al., 2006; Craven et al., 2006; Craven & Biffin 2010). Waterhousea (B.Hyland)

59 Craven & Biffin, comb. nov. is now considered a section in subgenus Acmena (DC.) Craven

60 & Biffin, comb. nov., in the genus Syzygium.

62 Anatomical and micromorphological characters have shown to be relevant for taxonomic

63 delimitation in the family (Schmid, 1980; Fontenelle et al., 1994), but only a few species

64 have been examined. The leaf anatomy of a number of species of Syzygium, but not S.

65 floribundum, was investigated by Soh & Parnell (2011) and shown to be taxonomically

66 useful for the infrageneric classification of the genus. The leaf surface micromorphology has

67 not been described for any species in the genus. This investigation aims to undertake a

68 complete anatomical and micromorphological description of the leaves of Syzygium

69 floribundum so as to determine if there are any taxonomically important characters that

70 differentiate this species from others in the genus.

72 MATERIAL AND METHODS

74 SAMPLING

75 Mature leaves of Syzygium floribundum were collected as fresh material from the natural

76 habitat of the species (FIG 1 B). Leaves were collected from sun-exposed branches from

77 randomly selected individual trees in a riverside forest in Enoggera, QLD (27° 26' 27" S /

78 153° 0' 45" E) and a small rainforest 2 km away from the coast in Noosa, QLD (26° 38' 52" S

79 / 153° 2' 36" E). Specimens (Reta-056.1, Reta-056.2, Reta-056.3 and Reta-056.4) are

80 currently housed at the Queensland Herbarium (BRI). The location of sampling localities and

81 the geographical distribution of the species are shown in the FIG 2.

83 SCANNING ELECTRON MICROSCOPY

84 Leaves were fixed in FAA for 24-48h, dehydrated using a graded ethanol series and then

85 critical point dried (Anderson, 1951) in an Autosamdri-815 automatic critical point drier.

86 Samples were mounted on stubs with self-adhesive double-sided carbon discs and sputter-

87 coated with gold palladium for 175 sec using a Leica EM SCD005 Gold Coater. Examination

88 and photography were conducted using a FEI Quanta 200 SEM/ESEM operated at 10kV.

90 LIGHT MICROSCOPY

91 Leaves were fixed in FAA for 24-48h prior to being dehydrated through a graded ethanol

92 series and embedded in paraffin wax (Johansen, 1940; Ruzin, 1999). Transverse sections

93 (5µm thickness) were cut using a Leica rotary microtome. Staining of sections was performed

94 using a 0.05% (w/v) of ruthenium red in distilled water and a counterstaining with 0.1%

95 (w/v) solution of toluidine blue (TBO) in distilled water (O’Brien et al., 1964; Chaffey et al.,

96 2002). Leaf clearings were prepared by immersing tissue fragments of 1-2 cm2 in 10% KOH

97 at room temperature for 48 h followed by 7% NaClO for 2 h or until leaves turned transparent.

98 Cleared leaves were washed five times with distilled water, stained with 0.1% safranin and

99 mounted with lactoglycerol (lactic acid-glycerol 1:1). Sections were dried and mounted with

100 Entellan mounting medium. Slides were observed using a Nikon SMZ 800 Stereo light

101 microscope (Nikon eclipse 50i compound) and images captured using the NIS Elemental

102 digital image analysis software.

103

104 TERMINOLOGY

105 Terminology for general anatomical descriptions, including glands, stomatal complex types

106 and cell types were based on Esau (1953) and Gifford and Foster (1989). Myrtaceae-specific

107 terminology was based on previous work on this family (Schmid, 1980, 1984; Keating, 1984;

108 Cardoso et al., 2009; Soh & Parnell, 2011 and da Silva et al., 2012).

109

110 RESULTS

111 GROSS MORPHOLOGY OF LEAVES

112 The leaves of S. floribundum are simple, opposite, lanceolate-elliptical in shape. The apex is

113 acuminate and the base attenuate. Both surfaces are punctuated. Leaves have numerous

114 secondary and tertiary reticulate veins. Venation is pinnate and weakly to strongly

115 brochidodromus. Intramarginal veins are visible on both adaxial and abaxial surfaces (FIG. 3

116 A).

117

118 CUTICLE, EPIDERMAL CELLS AND STOMATA

119 The adaxial cuticle is thicker than the abaxial cuticle. The cuticle contains polyphenols as

120 indicated by bluish-green staining with TBO. Epicuticular waxes are mainly granules and

121 flakes and randomly distributed on the surface (FIG. 3 B).

122 Adaxial and abaxial epidermal cells are small, compressed, plano-convex and mainly

123 isodiametric. Adaxial epidermal cells are rounded and have straight cell walls, while abaxial

124 epidermal cells are irregularly rounded and the anticlinal cell walls are strongly sinuous (FIG.

125 3 C).

126 Stomatal complexes are cyclo-staurocytic and randomly distributed on the abaxial surface of

127 the leaf (Fig. 3 B), but also can occur in groups above midrib. Guard cells are kidney-shaped

128 and the pore between them is narrow at the equatorial region. Stomata are circular to

129 elliptical and are located at the same level of the epidermal cells. The length of the guard

130 cells is 8–13 m long.

131

132 MIDRIB

133 The midrib of S. floribundum is a mixture of closed and complex open, with two or more

134 vascular bundles packed in the centre of the leaf (FIG. 4 A, B). The leaf blade is impressed

135 on the adaxial surface above the midrib. The bundle sheath extension is composed of

136 rounded-polygonal cells and is clearly developed, particularly on the abaxial side (FIG. 4 A).

137 The midrib is arc-shaped with a deep curvature. The abaxial side is convex and the adaxial is

138 slightly concave to straight (FIG. 4 B). Fibres are continuous around the midrib. Xylem

139 vessels of the midrib show scalariform perforation plates and helical wall thickenings. This

140 species has internal phloem, with a well-developed adaxial phloem partition. Incurved

141 margins of the phloem are poorly developed. Phloem sieve tubes and companion cells have

142 thin primary cell walls. Phloem fibres have evident and thick secondary cell walls.

143

144 MESOPHYLL

145 The mesophyll is dorsiventral and is formed by a 1-2 layered palisade parenchyma and a

146 spongy parenchyma with abundant intercellular spaces. The palisade parenchyma layer is

147 somewhat loose and composed of rectangular, attenuated and vertical cells. These cells

148 possess thin primary cell walls and numerous chloroplasts. The spongy parenchyma is

149 composed of irregular shaped cells (rounded to star-shaped). Both palisade and spongy

150 parenchyma contain mucilage (FIG 4 C). Subepidermal idioblasts with rhombus-shaped

151 calcium oxalate crystals occur throughout the palisade parenchyma (FIG. 4 D). Secretory

152 cavities are present on both sides of the leaf, are abundant throughout the mesophyll and have

153 variable dimensions (62 ± 25 m of diameter). They are composed of large spaces

154 surrounded by a sheath of peripheral epithelial cells. These cells are almost completely

155 disintegrated in mature leaves (FIG. 4 E). In paradermal view, secretory cavities are observed

156 as one overlying cell surrounded for 6-8 elongated cells. There are no stomata in the vicinity

157 of the secretory cavities (FIG. 3 D).

158

159 DISCUSSION

160 Syzygium floribundum shares a number of anatomical and micromorphological characters

161 with other Myrtaceae. Some of these features include the presence of calcium oxalate crystals,

162 internal phloem and schizogenous secretory cavities. Anatomical characters described here

163 largely agree with Keating (1984) and Soh & Parnell (2011) for Syzygium/Waterhousea.

164 Nonetheless, the combination of some features such as cyclo-staurocytic stomata, rhombus-

165 shaped crystals, midrib with a well-developed adaxial phloem partition and poorly developed

166 incurved margins of the phloem are not found in other species of the genus.

167

168 Calcium oxalate crystals have been reported in practically all the Myrtaceae studied (Cardoso

169 et al., 2009; Gomes et al., 2009; Soh & Parnell, 2011). However, rhombus-shaped calcium

170 oxalate crystals as those found in this study have been reported in a few Myrtaceae, mainly

171 some species of Syzygium (Soh & Parnell, 2011). The function of these crystals is not

172 completely clear, but has been related to the regulation of calcium and other minerals (Volk

173 et al., 2002), as well as protection against herbivores and pathogens (Franceschi & Nakata,

174 2005; Korth et al., 2006).

175

176 Internal phloem was found in all vascular bundles of leaves, either as continuous tissue or as

177 different degrees of adaxial phloem partition. This feature is regarded as a typical anatomical

178 character in the order Myrtales (Takhtajan, 1980; Cronquist. 1981) and is widely present in

179 Myrtaceae (Schmid, 1980; Cardoso et al., 2009). Further investigation is needed to determine

180 whether the internal phloem is derived from the procambium, or from mesophyll cells

181 (ground tissue). This developmental difference is regarded as a potentially informative

182 taxonomic character (Patil et al., 2009).

183

184 Secretory cavities follow the typical schizogenous pattern commonly observed in Myrtaceae

185 (Alves et al., 2008; Donato & Morretes, 2009; Gomes et al., 2009). The schizogenous pattern

186 can be identified by the presence of elongated epithelial cells surrounding the secretory space

187 in mature leaves (Cicarelli et al. 2008). Other patterns identified in Myrtaceae include

188 schizolysigenous secretory cavities (a mixture of schizogenous and lysigenous, the latter due

189 to disintegration of cells) in Myrtus communis (Cicarelli et al. 2008). Secretory cavities are

190 the structures referred by Hyland (1983) as oil dots on the surface of the leaves, which are

191 regarded as very numerous. Volatile oils secreted by these structures in Myrtaceae, have been

192 identified as flavonoids (Wollenweber et al., 2000) and terpenoids (Tanaka et al., 1996; Lee,

193 1998; Judd et al., 1999). Chemical compounds produced by secretory cavities were not

194 characterized in this study.

195

196 Leaf anatomical characters are generally regarded as homoplaseous in the genus Syzygium.

197 However some have been shown to be useful for species identification, such as shape of the

198 midrib, type of crystals and stomatal complexes (Soh & Parnell, 2011). Anatomical

199 characters described here are similar to those described by Soh and Parnell (2011) in the

200 subgenus Acmena (DC.) Craven & Biffin, where the species previously treated as

201 Waterhousea are included. In ecological terms, a thin cuticle, a loose palisade parenchyma

202 and superficial stomata suggest a mesophytic anatomy of the leaves (Esau, 1959). This

203 assumption matches with the common habitats of S. floribundum, which are mainly

204 rainforests, creeks and other riparian environments (Hyland, 1983; Wilson, 2011).

205

206 In this study, the leaf micromorphology and anatomy of Syzygium floribundum has been

207 described for the first time. There are anatomical similarities between the species and other

208 Myrtaceae taxa, particularly in terms of typical characters of the family. Certain anatomical

209 characters are coincident with other species previously named as Waterhousea, which

210 supports the grouping of these species. Combination of anatomical characters is unique for

211 this taxon within the genus, which is relevant for identification purposes. More anatomical

212 and micromorphological studies in the group could enhance the understanding of the

213 ecophysiology of species of Myrtaceae occurring in Australasian rainforests.

214

215 ACKNOWLEDGEMENTS

216 We are very grateful to Amy Carmichael, Rachel Hancock and technician staff from QUT for

217 valuable help and technical advice. Thanks to members of the Plant Structure and

218 Systematics group at QUT for feedback and advice. Funding for this investigation has been

219 provided for CONICYT-Government of Chile and QUT.

220

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342 AUTHOR INFORMATION

343 Hernan Retamales is PhD candidate at QUT working on the systematics of Australasian and

344 South American Myrtaceae using molecular and morpho-anatomical data. His PhD is funded

345 by the Government of Chile (CONICTY). Dr Rosa Scherson is a Lecturer at the School of

346 Forestry in the University of Chile and is the external supervisor of Mr Retamales. Dr Tanya

347 Scharaschkin is a Senior Lecturer at the School of Earth, Environmental and Biological

348 Sciences at QUT. She has recused herself from the review and editorial process for this

349 article, due to conflict of interest as Editor of PRSQ, which has been handled by Dr. Geoff

350 Edwards (President RSQ) instead.

351

352

353 FIG. 1. Syzygium floribundum, habit and leaf morphology. A, Tree. B, Detail of leaves.

354

355

356 FIG 2. Geographical distribution of S. floribundum showing sampling localities. Red dots-

357 geographical distribution of the species. Green dots- sampling localities. Map obtained and

358 adapted from: Australia’s Virtual Herbarium (2014).

359

360 FIG. 3.

361 Light (LM) and scanning electron micrographs (SEM) of leaves of S. floribundum. A, Leaf

362 clearing showing reticulate tertiary venation. B, SEM micrograph showing distribution of

363 stomata on the leaf surface. C, SEM micrograph showing detail of cyclo-staurocytic stomata

364 and surrounding epidermal cells. D, SEM micrograph of secretory cavity on the abaxial

365 surface showing overlying cell (arrow) surrounded by 6-8 elongated cells. Scale bars = 500

366 µm in A, 20 µm in C.

367

368

369

370

371

372

373

374

375 FIG. 4. Transverse light micrographs (LM) of leaves of S. floribundum. A, General view of

376 the anatomy of the leaf blade showing slight depression above the midrib. B, Detail of the

377 midrib showing xylem (stained green-blue), phloem (pink) and fibres (purple). C,

378 Dorsiventral mesophyll showing palisade and spongy parenchyma. D, Detail of the

379 mesophyll containing subepidermal idioblasts with rhombus-shaped calcium oxalate crystals.

380 E, Secretory cavity composed of a large space surrounded by epithelial cells. cr- calcium

381 oxalate crystals, fi- fibres, ph- phloem, pp- palisade parenchyma, sc- secretory cavities, sp-

382 spongy parenchyma, xy- xylem. Scale bars = 100 µm