beni-suef university journal of basic and applied sciences 2 (2013) 120e127 Available online at www.sciencedirect.com ScienceDirect journal homepage: www.elsevier.com/locate/bjbas Full Length Article A contribution to the specification of Caesalpinioideae (L) based on morphological and molecular criteria Usama K. Abdel-Hameed*, Usama I. El-Magly, Ishak F. Ishak, Mohamed E. Tantawy Ain Shams University, Faculty of Science, Botany Department, Abassia, Cairo, Egypt article info abstract Article history: The present study included the investigation of both morphological attributes of some taxa Received 10 January 2013 of Caesalpinioideae viz. whole plant, leaf architecture & epidermal characteristics (LM & Accepted 15 March 2013 SEM) and certain molecular attributes (RAPD & Isozymes) to clarify the diversity and the Available online 1 November 2013 diagnostic importance of these characters. The sum of both character states of morpho- logical and molecular criteria (326 attributes & 353 bands) respectively of total (679 attributes) Keywords: of the investigated taxa were subjected to a numerical analysis using NTsys-pc program Morphology (version 2.02). The resulted dendrogram interpreted the similarities and dissimilarities be- Leaf architecture tween the investigated taxa. The specific relationships were discussed and compared with Stomatography some of the current systems of classification. The aim of the present study is tried to find the Biochemical markers interspecific relationships of the studied taxa through the investigation of their morpho- Caesalpinioideae logical and molecular characters in addition to a numerical evaluation of such characters. Among the reached concluding remarks, The dendrogram resulted from morphological and molecular attributes supported the separation of Cassia and Senna as two taxonomic entities. Copyright 2013, Beni-Suef University. Production and hosting by Elsevier B.V. All rights reserved. 1. Introduction The principal characteristics of the leaf venation pattern of a species are genetically fixed. This provides the basis for Caesalpinioideae includes 171 genera and about 2250 species using the leaf venation as a taxonomic tool (Hickey, 1973; of tropical and sub-tropical trees and shrubs (Lewis et al., Roth-Nebelsick et al., 2001). Seetharam and Kotresha (1998) 2005). Boulos (1999) recorded the following wild species in emphasized the taxonomic importance of venation and its Egyptian flora viz. Cassia italica, Cassia holosericea, Cassia occi- usefulness in classification of Bauhinia L. dentalis, Cassia senna and Delonix elata. * Corresponding author. E-mail addresses: [email protected] (U.K. Abdel-Hameed), [email protected] (M.E. Tantawy). Peer review under the responsibility of Beni-Suef University Production and hosting by Elsevier 2314-8535/$ e see front matter Copyright 2013, Beni-Suef University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bjbas.2013.03.004 beni-suef university journal of basic and applied sciences 2 (2013) 120e127 121 Leaf epidermal studies have shown that stomata can pro- Table 1 e Collection data of Caesalpinioideae. vide valuable taxonomic and systematic evidence in both living and fossil plants and also have played a significant role No. Taxa Source in framing hypotheses about early angiosperm evolution 01 Bauhinia alba Buch.-Ham. ex Wall. OBG (Carpenter, 2005). Zou et al. (2008) examined the leaf 02 B. hookeri F. Muell. BGC epidermal micro characters of nine taxa of Cercis using SEM & 03 B. variegata L. BGA LM, and then concluded that the interspecific differences are 04 Brownea grandiceps Jacq. ZBG 05 Caesalpinia ferrea Tulasne. ZBG minor in the genus. 06 C. gilliesii (Wallich ex Hook.) Dietr. BGC RAPD markers are powerful techniques for determining 07 Cassia fistula L. OBG intra- and interspecific genetic variations and allow direct 08 C. grandis L.f. ZBG comparison of plant variation at both biochemical and mo- 09 C. javanica L. ZBG lecular levels (Williams et al., 1990; Welsh and McClelland, 10 C. marginata Roxb. OBG 1990; Carlier et al., 2004). RAPD markers have been reported 11 C. nodosa Buch-Ham. ex Roxb. OBG to be as efficient as AFLP, SSR, RFLP and ISSR markers (Martins 12 Ceratonia siliqua L. BGA 13 Cercis chinensis Bunge OBG et al., 2003; Zahuang et al., 2004) for genetic analysis at 14 Delonix regia (Bojer ex Hook.) Raf. BGA different plant species. 15 Gleditsia caspica Desf. OBG Whitty et al. (1994) adopted RAPD method for use as a 16 Haematoxylum campechianum L. BGA phenetic tool on the legume tribe Cassiinae, using eight 17 Parkinsonia aculeata L. BGA primers and showed the potential for separation of the 18 Peltophorum africanum Sond. BGA nodulated nitrogen fixing genus Chamaecrista from the previ- 19 Saraca indica L. OBG ously congeneric groups Cassia and Senna. Diallo et al. (2007) 20 Schotia brachypetala Sond. OBG 21 Senna alata (L.) Roxb. BGC studied 10 Tamarindus populations using markers RAPDs, the 22 S. didymobotrya (Fres.) Irwin & Barneby BGA results showed that Tamarindus indica has a high intra popu- 23 S. sophera (L.) Roxb. ZBG lation genetic variability. 24 S. surattensis (Burm. f.) Irwin & Barneby ZBG Despite the use of DNA markers, isozymes are still widely 25 Tamarindus indica L. OBG employed in species delimitation, conservation and cultivar BGA: Botanical Garden, Ain Shams University, Faculty of Science, identification (Samec et al., 1998; Mohamed, 2006). Isozymes Cairo, Egypt. BGC: Botanical Garden, Cairo University, Faculty of have been applied as molecular-genetic markers to study ge- Agriculture, Giza, Egypt. OBG: Orman Botanical Garden, Ministry of netic diversity and phylogenetic affinities in populations of Agriculture, Giza, Egypt. ZBG: Zohria Botanical Garden, Ministry of Gleditsia triacanthos (Schnabel and Hamrick, 1990), Cassia Agriculture, Gezzera, Cairo, Egypt. species (Nualkaew et al., 1998; Siva and Krishnamurthy, 2005). Concerning numerical analysis, several authors checked the Macromorphological attributes of the whole plant were current classification for different genera and species of Legu- described from the investigated specimens or compiled from minosae and analysed their results by using different numerical text books viz. Bailey (1949). analysis programs. Larmarque and Fortunato (2003) used the Lamina vein architecture was carried out according to the numerical analysis to discuss the taxonomic placement of Acacia customary method of Jesudass et al. (2003). Leaf architectural emiliona and its affinity within subgenus Aculeiferum. Tantawy terminology generally follows Hickey (1973) and LAWG (1999). et al. (2005) showed the similarities between some of different Stomatography was carried on the bases of traditional taxa of Mimosoideae. El-Gazzar et al. (2008) reached to computer- method of Stace (1965). The photomicrographs were taken generated keys to the flora of Egypt (Mimosoideae & Caesalpi- using a Reichert Microstar IV microscope at the Plant Taxonomy nioideae). Abou El-Enain et al. (2007) delimited the genus Cassia Research Laboratory, Botany Department, Faculty of Science, into two subgenera viz. Fistula and Senna based on the basis of Ain Shams University, Cairo, Egypt. For SEM small pieces morphological criteria and seed protein electrophoresis. (7 mm2) of the leaf material were fixed on SEM stubs with The aim of the present study is tried to find the interspe- double-sided tape, coated with gold in SPI-Module sputter cific relationships of the studied taxa through the investiga- coater, examined and photographed in Jeol JSM 5200 at different tion of their morphological and molecular characters in magnifications ranged from 750 X-1500X. Descriptive addition to a numerical evaluation of such characters. Table 2 e Primers used in RAPD analysis. 2. Materials and methods No. Primer Sequence 1 SC10-5 TCGGAGTGGC Fresh mature leaf materials of 25 caesalpinioid taxa grown in 2 SC10-14 TCCCGACCTC some Egyptian botanical gardens were collected and subjected 3 SC10-17 GTTAGCGGCG for the present study (Table 1). Identification was confirmed 4 SC10-18 GCCCTACGCG according to (Bailey, 1949; Bircher, 1960). 5 SC10-22 CTAGGCGTCG The taxa were further matched against dried specimens in 6 SC10-23 GGCTCGTACC the Herbaria of Ain Shams University (CAIA), Cairo University 7 SC10-25 CGGAGAGTAC (CAI), Flora & Phytotaxonomy & Agriculture Research Center 8 SC10-59 GCATGGAGCT 9 SC10-64 CCAGGCGCAA (CAIM) and Orman Botanical Garden. Voucher specimens of 10 SC11-30 CCGAAGCCCT the studied taxa are deposited in CAIA. 122 beni-suef university journal of basic and applied sciences 2 (2013) 120e127 terminology of epidermal characteristics based on Metcalfe and carried out according to Whitty et al. (1994) and the primers Chalk (1950), Murley (1951), LAWG (1999) and Prabhakar (2004). used are presented in Table 2. Genomic DNA extraction was performed as suggested by The utilized isozymes are a- and b-esterase (a- and b- Est), DNA extraction kit’s manufacturer Jena Biosciences, Plant acid phosphatase (Acph), alcohol dehydrogenase (Adh), and DNA Preparation Kit. Polymerase chain reactions (PCR) were aldehyde oxidase (Alo). These isozymes were separated in 10% Fig. 1 e A&B, Major primary vein categories of lamina architecture; A, Campylodromous. B, Pinnate. C-E, Major secondary vein categories of lamina architecture; C, Brochidodromous. D, Festooned brochidodromous. E, Cladodromous. F-J, Major types of stomata; F, Paracytic. G, Isotricytic. H, Tetracytic. I, Anomocytic. J, Cyclocytic. KeO,