Bangladesh J. Plant Taxon. 29(2): 283-296, 2022 (December) DOI: https://doi.org/10.3329/bjpt.v29i2.63530 © 2022 Bangladesh Association of Plant Taxonomists MACROMORPHOLOGICAL, ANATOMICAL AND MOLECULAR STUDIES OF SOME TAXA OF ARALIACEAE IN EGYPT MAI M WAHBA*, ASHRAF S HAIDER1, MAGDY M MOURAD2, IA MASHALY AND IHSAN E EL-HABASHY Botany Department, Faculty of Science, Mansoura University, Egypt Keywords: Morphology; Anatomical structure; Lamina architecture; Stomatography; ISSR, Araliaceae. Abstract The present study investigated morphological features, leaf and stem anatomy, leaf architecture, epidermal characteristics, and molecular characters of some taxa of Araliaceae to trace out the diversity and the diagnostic significance of these attributes. The studied taxa based on combination of 260 characters representing 182 morphological and 78 molecular characters which were subjected to a numerical analysis using NTSYS- PC program. The generated dendrogram explained the similarities and the differences between the examined taxa. The specific similarities are discussed and compared with some current classification systems. The generated dendrogram from morphological attributes confirmed the separation of Aralieae and Schefflerieae as two tribes of Araliaceae and supported the separation of simple leaved taxa from compound leaved. Introduction Araliaceae comprises 47 genera and over 1.350 species (Wen et al., 2001) Five of the six largest genera with 50 or more species (Schefflera J.R.Forst., Oreopanax Decne. & Planch, Dendropanax Decne. & Planch., Polyscias J.R.Forst. and Osmoxylon Miq.) are best represented in tropical or subtropical zones although several smaller genera (Brassaiopsis Decne. & Planch, Panax L., Macropanax Miq., Hedera L., Oplopanax Torr. & A.Gray, and Gamblea C.B.Clarke) are found in the North Temperate Zone. Araliaceae are also well-developed in the Old Worldin southeastern Asia, the Pacific, and Indian Ocean basins. New World araliads include only a few genera, most of them are also largely the Old World such as Aralia, Oplopanax, Panax, Pseudopanax K.Koch, and Dendropanax. After the inclusion of Sciadodendron Griseb., in Aralia by Wen (2002), Oreopanax is now the only genus in the New World. Araliaceae trees or shrubs, sometimes woody vines. Leaves simple, palmately compound or 1-3 pinnately compound, lobed. Fruits drupe or berry. A significant step was taken in resolving the placement of Araliaceae among the main genealogy of the order Apiales (Plunkett et al., 2004; Plunkett et al., 2001a) and in knowing the relationships within and between related genera of Araliaceae (EIBL et al., 2001; Plunkett et al., 2001b; Wen et al., 1996). Harms (1894–1897) classified the family into three tribes. The tribe Aralieae with imbricate aestivation and Mackinlayeae and Schefflereae with valvate aestivation are separated from one another by petal insertion based on petal aestivation and base insertion. Bentham (1867) provided nearly similar tribes Mackinlayeae and Aralieae, but the genera that placed in Schefflereae by (Harms, 1894–1897) were treated as tribes Panaceae and Hedereae (with smooth or ruminate endosperm, respectively), in addition to Plerandreae (where stamen number exceeded petal number). *Correspondent author: E-mail: maiwahba18@gmail.com 1Botany Department, Faculty of Science, Tanta University, Egypt. 2Botany Department, Faculty of Science, Ain Shams University, Egypt. https://doi.org/10.3329/bjpt.v29i2.63530 mailto:maiwahba18@gmail.com 284 WAHBA et al. Based on morphological features by Harms (1898) and Judd et al., (1994) and anatomical evidence by Metcalfe and Chalk-Vol (1950), Araliaceae have been put with Apiaceae which is supported by the recent molecular studies (Plunkett et al., 1996; Plunkett et al., 1997). Jacobs et al. (2010) studied fruit set in Hedera helix. Mourad (2013) showed the separation of simple leaved Meryta denhamii from lobed (Hedera helix and Tetrapanax papyrifer) and compound leaved Polyscias spp. Amini et al. (2020) studied the micromorphology of Hedera species in Iran. Lestari and Elya (2019) made macroscopic studies of Polyscias guilfoylei leaves. The essential use of leaf architectural character as an aid in the delimitation of genera and species was performed in paleobotany (Dilcher, 1974; Mouton, 1966). Zhernova et al. (2021) made comparative wood for the anatomy of Astropanax Seem., and Neocussonia (Harms) Hutch. Săvulescu and Luchian (2009) studied the diagnostic value of Hedera epidermis and epidermis that is made up of one cell layer with polygonal cells and thin lateral wall. studied epidermal cell descriptions of Hedera species in Iran. Kotina et al. (2010) surveyed the bark anatomy of Araliaceae and some related taxa. Ostroumova et al. (2010) surveyed the leaf anatomy of Araliaceae and some related taxa. Rout et al. (2007) used RAPD and ISSR markers to study the genetic relationship between Polyscias and Schefflereae. Hoi et al. (2021) used Inter Simple Sequence Repeat (ISSR) markers to assess the genetic diversity of Panax bipinnatifidus. Araliaceae has a taxonomic problem within and between its related genera. Aralieae and Schefflerieae were not accurately delimited and leaf forms were represented within the family of Araliaceae that have a tremendous array. This study aims to try to find the interspecific similarities of the studied taxa by investigating their morphological, anatomical, and molecular characteristics as well as a numerical evaluation of such traits. Materials and Methods Sampling Twelve taxa of Araliaceae representing six genera were collected from the Botanical Garden of Mansoura University and Orman Botanical Garden, Giza, Egypt (Table 1). Identification was confirmed by comparison with herbarium specimens in the herbarium of Ain Shams University, Faculty of Science (CAIA). Voucher specimens of the investigated species were kept in Mansoura herbarium, Botany Department, Faculty of Science, Mansoura University. Nomenclature has been updated according to several websites (https://www.ipni.org/). Macro-micromorphological investigations Macromorphological characters of the leaves, inflorescence, flowers, and fruits were described from the fresh specimens. For the anatomical features, the methods were characterized by Johansen (1940) and were adopted by Jensen (1962) and Peacock (1973). Leaf vein architecture was performed according to the usual method of(JESUDASS et al., 2003). Laminaʹs architectural terminology follows (Ash, 1999; Hickey, 1973). Stomatography was performed according to the method of Stace (1965). By using a Reichert Microstar IV microscope, the photomicrographs were taken at the Plant Taxonomy Research Laboratory, Botany Department, Faculty of Science, Ain Shams University, Cairo, Egypt. For scanning electron microscope (SEM) small (7 mm2) pieces of the lamina, the material was installed on SEM stubs with double-sided tape, coated with gold in SPI-Module sputter coater, checked, and photographed in Jeol JSM 5200 at various magnifications (500x, 1000x). The description of epidermal characteristics terminology based on (Ash, 1999; Metcalfe and Chalk- Vol, 1950; Murley, 1951; Prabhakar, 2004). https://www.ipni.org/). MACROMORPHOLOGICAL, ANATOMICAL AND MOLECULAR STUDIES 285 Table 1. List of the studied Araliaceae taxa and their collection data. Location Date of Collection Taxa No. Mansoura University Garden 5/2020 3/2021 Hedera canariensis Willd., Mag. Neuesten Entdeck. Gesammten Naturk. Ges. Naturf. Freunde Berlin 2: 171 (1808). Synonym: H. grandifolia Hibberd, The Ivy 96 (1872). 1 // 5/2020 3/2021 H. helix L., Sp. Pl. 1: 202 (1753). Syn: H. donerailensis Hort. ex K.Koch, Dendrologie 1: 680 (1869). 2 Orman Botanical Garden 5/2020 4/2021 Meryta denhamii Seem., Bonplandia 10: 295 (1862). Syn: M. macrocarpa Baill., Adansonia 12: 155 (1878). 3 // 6/2020 2/2021 Oreopanax guatemalensis Decne. & Planch., Rev. Hort. [Paris]. Ser. IV, iii. (1854) 108, nomen. Syn: O. obtusifolius L.O.Williams, Fieldiana, Bot. 31: 20 (1965). 4 // 5/2020 3/2021 Polyscias fruticosa Harms, Nat. Pflanzenfam. [Engler & Prantl] iii. (1894) 45. Syn: Aralia tripinnata Blanco, Fl. Filip. [F.M. Blanco] 223 (1837). 5 Mansoura University Garden 5/2020 3/2021 P. guilfoylei L.H.Bailey, Rhodora 1916, xviii. 153. Syn: Aralia guilfoylei W.Bull, Cat. New Beautiful Rare Pl. [W. Bull] 83: 4 (1873). 6 // 5/2020 3/2021 P. scutellaria (Burm.f.) Fosberg, Occas. Pap. Univ. Hawaii 46: 9 (1948). Syn: Aralia cochleata Lam., Encycl. [J. Lamarck & al.] 1(1): 224 (1783). 7 // 5/2020 3/2021 Schefflera actinophylla (Endl.) Harms, Nat. Pflanzenfam. [Engler & Prantl] 3(Abt. 8): 36 (1894). Syn: Brassaia singaporensis Ridl., J. Straits Branch Roy. Asiat. Soc. 75: 38 (1917). 8 // 5/2020 3/2021 S. arboricola (Hayata) Hayata ex Merr., Lingnan Sci. J. 5(1-2): 139 (1928). Syn: Heptapleurum arboricola Hayata, Icon. Pl. Formosan. 6: 23 (1916). 9 Orman Botanical Garden 6/2020 2/2021 S. elegantissima (Veitch ex Masters) Lowry & Frodin, Baileya 23(1): 9 (1989): (1989). Syn: Schefflera fagueti Baill., Adansonia 12: 142 (1878). 10 Mansoura University Garden 5/2020 3/2021 S. pueckleri (K.Koch) Frodin, Baileya 23(1): 10 (1989). Syn: Tupidanthus calyptratus Hook.f. & Thomson, Bot. Mag. 82: t. 4908 (1856). 11 Orman Botanical Garden 6/2020 2/2021 Tetrapanax papyrifer (Hook.) K.Koch, Wochenschr. Gärtnerei Pflanzenk. 2: 371 (1859). Syn: Aralia mairei H.Lév., Repert. Spec. Nov. Regni Veg. 13: 342 (1914). 12 286 WAHBA et al. Molecular assessment (ISSR-PCR analysis) Genomic DNA was extracted from the twelve samples according to the manufacturer protocol of the Gene JET Genomic DNA Purification Kit (K0721/ Thermo fisher). Total genomic DNA was amplified through Gene Amp Polymerase Chain Reaction (PCR) system cycler. PCR for amplified genomic DNA was carried out according to(El-Assal et al., 2011). ISSR-PCR reactions were conducted using 6 primers for the genotype (Table 2). Gel documentation system (Geldoc-it, UVP, and England), was applied for data analysis using Totallab analysis software (Ver.1.0.1), ww.totallab.com. Table 2.ISSR primers names and sequence. No Primers Sequences 1 iPBS primer 2270 5´-ACCTGGCGTGCCA-3´ 2 C1 5´-AGGGCTGGAGGAGGGC-3´ 3 G4 5´-ACTGACTGACTGACTG-3´ 4 PseCra5 F-5´-CCAGCGTCACCTCCATTATT-3´ R-5´-TCACAGCCAGCCACTGTATC-3´ 5 PseLes1 F-5´-AAGTTGATGGCTTCGCTCAT-3´ R-5´-ACCACCCCAATACAAAACCA-3´ 6 PseCra3B F-5´-ATGTTTGTGAATTGTGAGTGTGG-3´ R-5´-CCCCATCTTTTGTCCCTCA-3´ Data analysis The UPGMA function and SAHN program were used by Sneath and Sokal (1973). All computations were made with the help of NTSYS-PC version 2.02 (Rohlf, 1998). Results and Discussion Shape of leaves Simple in Hedera canariensis, Meryta denhamii, and Oreopanax guatemalensis, lobed palmate in Hedera helix, and Tetrapanax papyrifer, compound palmate in 4 species of genus Schefflera and compound pinnate in 3species of genus Polyscias are as shown in Fig. 1. Stem and lamina anatomy Stem investigations Stem angled in seven taxa viz., Hedera canariensis, Meryta denhamii, Oreopanax guatemalensis, Polyscias fruticosa, Schefflera arboricola, Schefflera elegantissima, Tetrapanax papyrifer and terete in five taxa Hedera helix, Polyscias guilfoylei, Polyscias scutellaria, Schefflera actinophylla and Schefflera pueckleri. All taxa are not glandular except Hedera canariensis. Lenticel present in six taxa viz., Hedera helix, Meryta denhamii, Oreopanax guatemalensis, Schefflera elegantissima, Schefflera pueckleri and Tetrapanax papyrifer, but absence in other six taxa, collenchyma may be angular-lamellar in nine taxa, and angular in Meryta denhamii, Oreopanax guatemalensis and Schefflera arboricola. The aspect of vascular bundles is Siphonostelic in 11 taxa and distinct in Polyscias scutellaria as observed in Fig. 2. MACROMORPHOLOGICAL, ANATOMICAL AND MOLECULAR STUDIES 287 Fig. 1. (A-D) Leaves photographs of some studied taxa; A) Simple; B) Simple lobed palmate; C) Compound pinnate; D) Compound palmate. Lamina anatomy Raised adaxially in 11 taxa and flattened adaxially in Schefflera actinophylla happen. All taxa are not glandular except in Hedera helix is peltate eglandular, while Tetrapanax papyrifer is multicellular branched eglandular. Collenchyma annular in five taxa Hedera canariensis, Hedera helix, Polyscias fruticosa, Schefflera actinophylla, Schefflera elegantissima. In addition, annular- lamellar is in 5 taxa Meryta denhamii, Oreopanax guatemalensis, Schefflera arboricola, Schefflera pueckleri and Tetrapanax papyrifer, angular-lamellar in Polyscias guilfoylei and angular in Polyscias scutellaria. Vascular system partially continuous is in 6 taxa Hedera canariensis, Hedera helix, Oreopanax guatemalensis, Polyscias fruticosa, Polyscias guilfoylei and Schefflera actinophylla and distinct in other six taxa. All taxa have druses-raphides except druses in Polyscias scutellaria (Fig. 2). Lamina vein architecture Primary vein pinnate in six taxa viz., Meryta denhamii, Oreopanax guatemalensis, Polyscias fruticosa, Schefflera actinophylla, Schefflera elegantissima and Schefflera pueckleri, suprabasal in Hedera canariensis, Hedera helix, acrodromous (basal) in Polyscias guilfoylei, suprabasal actinodromous in Polyscias scutellaria, suprabasal actrodromous in Schefflera arboricola, palinactinodromous in Tetrapanax papyrifer. Secondary vein brochidodromousis in four taxa, namely, Hedera canariensis, Hedera helix, Polyscias guilfoylei, and Polyscias scutellaria, 288 WAHBA et al. reticulodromous in Meryta denhamii, Schefflera arboricola, festooned brochidodromous in Oreopanax guatemalensis, Schefflera actinophylla, Schefflera pueckleri, weak brochidodromous in Polyscias fruticosa, intramarginal vein in Schefflera elegantissima, interior (seven basal veins) in Tetrapanax papyrifer. Third vein category random reticulate in seven taxa, alternate percurrent in four taxa, namely, Oreopanax guatemalensis, Polyscias fruticosa, Polyscias guilfoylei, and Polyscias scutellaria, dichotomizing in Schefflera elegantissima. 4° vein RPR (regular polygonal reticulate) in 9 taxa, alternate percurrent in Meryta denhamii, dichotomizing in Schefflera elegantissima, absence in Oreopanax guatemalensis. 5° category RPR in five taxa viz., Hedera canariensis, Hedera helix, Meryta denhamii, Schefflera arboricola and Tetrapanax papyrifer. Dichotomizing is in five taxa Polyscias guilfoylei, Polyscias scutellaria, Schefflera actinophylla, Schefflera elegantissima and Schefflera pueckleri, absence in Oreopanax guatemalensis, Polyscias fruticosa as Fig. 3. Fig. 2. (A-I) Photographs of some stem anatomy of studied taxa; A) Angled, egland unicellular unbranched trichome, siphonostelic vascular bundle; B) Terete, lenticel; C) Distinct vacular bundle; D) Angular collenchyma. E-I) Photographs of some lamina anatomy of studied taxa; E) Raised adaxially, peltate eglandular trichome, annular collenchyma, druses & raphides crystal, partially continuous vascular bundle; F) Druses crystal, angular collenchyma, distinct vascular bundle. G) Flattened adaxially; H) Multicellular branched eglandular trichome; I) Angular & lamellar. Abbreviations: Tr. trichome; Se. sub epidermal periderm; Vb. vascular bundle; Len. lenticel; AC. angular collenchyma; RC. raphides crystal; DC. druses crystal. Pc. palisade cells; RC. raphides crystal; Tr. trichome; AC. angular collenchyma. MACROMORPHOLOGICAL, ANATOMICAL AND MOLECULAR STUDIES 289 Fig. 3. (A-H) The main categories of lamina vein architecture with LM. A) Suprabasal 1°V, brochidodromous 2°V, random reticulate 3°V, regular polygonal reticulate 4°V 5°V; B) Pinnate 1°V, weak brochidodromous 2°V, alternate percurrent 3°V; C) Acrodromous 1°V, dichotomizing 5°V; D) Suprabasal actinodromous 1°V; E) Suprabasal acrodromous 1°V, reticulodromous 2°V; F) Dichotomizing 3°V, 4°V, 5°V. G) Festooned brochidodromous 2°V; H) Palinactinodromous 1°V. 290 WAHBA et al. Epidermal cell description Cell shape was irregular in 4 taxa, namely, Hedera canariensis, Hedera helix, Meryta denhamii, and Tetrapanax papyrifer and polygonal in the rest taxa. Anticlinal wall sinuous in four taxa Hedera canariensis, Hedera helix, Meryta denhamii, and Tetrapanax papyrifer and slightly curved in 8 taxa. Stomatal shape elliptical in all taxa. Stomatal type anomocytic and anisocytic is in Hedera canariensis and Hedera helix. Anisocytic in seven taxa, anisocytic and diacytic in Polyscias fruticosa, Polyscias guilfoylei and Schefflera elegantissima. Sculpture ruminate in four taxa Hedera canariensis, Oreopanax guatemalensis, Schefflera actinophylla. Schefflera pueckleri arepusticulate in Hedera helix and Meryta denhamii, reticulate-aerolate in Polyscias fruticosa, Polyscias guilfoylei, and Polyscias scutellaria reticulated in Schefflera arboricola, favulariate in Schefflera elegantissima and striate in Tetrapanax papyrifer (Fig. 4). Fig. 4. (A-C) Major categories of stomatography as revealed with LM; A) Anomocytic & anisocytic stomata, irregular cell shape, sinuous anticlinal wall; B) Anisocytic stomata, polygonal cell shape, slightly curved anticlinal wall; C) Anisocytic & diacytic. D-F) Major types of lamina surface sculpture with SEM; D) Ruminate; E) Pusticulate; F) Reticulate-aerolate. G-I) Major types of lamina surface sculpture with SEM; G) Reticulate; H) Favulariate; I) Striate. Molecular assessment All primers produced 78 monomorphic and polymorphic bands (Table 3). Primer iPBS primer 2270 produced one monomorphic band and 9 polymorphic bands (7 common and 2 unique), C1 produced one monomorphic band and 14 polymorphic bands (13 common and 1 unique), G4 produced one monomorphic band and 13 polymorphic bands (12 common and 1 unique), PseCra5 MACROMORPHOLOGICAL, ANATOMICAL AND MOLECULAR STUDIES 291 produced no monomorphic bands, and 13 polymorphic bands (13 commons). While no unique bands were produced, PseLes1 produced no monomorphic bands, and 13 polymorphic bands (13 commons). While no unique bands were produced, PseCra3Bproduced no monomorphic bands, and 13 polymorphic bands (13 commons), while no unique bands were recorded (Fig. 5). Table 3. Type of bands and percentage of polymorphism of ISSR primers applied on the studied taxa of family Araliaceae. Primer Monomorphic bands Polymorphic bands Total bands Polymorphism % Common Unique iPBS primer 2270 1 7 2 10 90 C1 1 13 1 15 93.33 G4 1 12 1 14 92.86 PseCra5 0 13 0 13 100 PseLes1 0 13 0 13 100 PseCra3B 0 13 0 13 100 Fig. 5 (A-F) ISSR profile of the studied taxa of Araliaceae generated by A) iPBS primer 2270; B) primer C1; C) primer G4; D) primer PseCra5; E) primer PseLes1; F) primer PseCra3B. 292 WAHBA et al. Numerical analysis The data obtained from the whole plant, stem, and leaf anatomy for the examined taxa were amalgamated with the data that was obtained from lamina architecture and stomatographic analyses. Then, they were subjected to numerical analysis to explain and discuss the similarity among the studied taxa based on (182) macro-micromorphological traits that were used for computation and produced dendrogram as shown in Fig. 6. The data extracted from ISSR analysis were subjected to numerical analysis to explain and discuss the similarity among the examined taxa based on (78) molecular traits. These traits were used for computation and produced dendrogram as shown in Fig. 7. Finally, the data extracted from macro-micromorphological attributes were amalgamated with the data from ISSR analysis. They were subjected to numerical analysis to explain and discuss the similarity among the studied taxa based on (260) macro- micromorphological and molecular traits that were used for computation and produced dendrogram as shown in Fig. 8. Fig. 6. Dendrogram of studied taxa of Araliaceae based on morphological characters (182). The resulting dendrogram from morphological attributes is compared with the current system treatments. The dendrogram shows that the taxa under investigation were splitted into two main series (I and II), three clusters (A, B, and C), and five groups (Fig. 6). Series I included only one cluster (A) and one group; Cluster A included one group of three studied species. Series II involved two clusters (B & C) and four groups and Cluster B involved two groups; the first group involved two studied species while the second one involved four studied species. Cluster C involved two groups; the first group involved two studied species when the second one involved only one studied species. The similarities among these taxa are summarized as follows. MACROMORPHOLOGICAL, ANATOMICAL AND MOLECULAR STUDIES 293 Fig. 7. Dendrogram of studied taxa of Araliaceae based on molecular characters (78). Fig. 8. Dendrogram of studied taxa of Araliaceae based on morphological and molecular characters (260). Series I, Group 1 includes Hedera canariensis, Hedera helix, Tetrapanax papyrifer. These results are in agreement with Harms (1894-1897 classification systems that put them in the same tribe (Hutchinson, 1967; Bentham, 1867; Chang-Jiang et al., 1982) placed them in different tribes. Calestani (1905) and Viguier (1906) placed Tetrapanax papyrifer in the same tribe, but Hedera canariensis, Hedera helix indifferent tribes. Seemann (1868) placed Tetrapanax papyrifer in the same family but different tribe and placed Hedera canariensis, Hedera helix in a different family. 294 WAHBA et al. Series II, Group 2 includes Meryta denhamii, Oreopanax guatemalensis. These results are in agreement with Harms (1894-1897) classification systems that put them in the same tribe. Hutchinson (1967), Bentham (1867), Chang-Jiang et al. (1982) and Seemann (1868) placed Meryta denhamii in the same tribe, but Oreopanax guatemalensis in a different tribe. Calestani (1905) and Viguier (1906) placed Oreopanax guatemalensis in the same tribe, but Meryta denhamiiin a different tribe. Group 3 includes Schefflera actinophylla, S. pueckleri, S elegantissima, S. arboricola. These results are in agreement with Harms (1894-1897), Calestani (1905) and Viguier (1906) classification systems that put them in the same tribe. Hutchinson (1967), Bentham (1867), Seemann (1868), Chang-Jiang et al. (1982) placed them in the same family but in different tribes. Group 4 includes Polyscias fruticosa, P. guilfoylei. These results are in agreement with Bentham (1867), Seemann (1868), Harms (1894-1897), Calestani (1905), Hutchinson (1967) and Chang-Jiang et al. (1982) classification systems that put them in the same tribe. Viguier (1906) placed it in the same family but different tribe. Group 5 includes Polyscias scutellaria. This result is in agreement with Bentham (1867); Seemann (1868); Harms (1894-1897); Calestani (1905); Hutchinson (1967) and Chang-Jiang et al. (1982) who put them in the same tribe. Viguier (1906) placed it in the same family but in a different tribe. 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