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Home , Bombacaceae, Bombacoideae, Bombax ceiba, Ceiba, Ceiba speciosa, Eriotheca

415 Advances in Environmental Biology, 7(2): 415-426, 2013 ISSN 1995-0756

This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE

Comparative study of three species of Malvatheca ( and ( sensu lato)using Morphological, Anatomical and RAPD-PCR analyses.

1Wafaa M. Said, 2Nahla O.M. Ehsan, 3Noha S. Khalifa

1,2Botany Dept., Faculty of Women for Arts, Science, and Education Ain Shams University. 3Botany Dept., Faculty of Science, Ain Shams University

Wafaa M. Said, Nahla O.M. Ehsan, Noha S. Khalifa: Comparative study of three species of Malvatheca (Bombacoideae and Malvoideae (Malvaceae sensu lato)using Morphological, Anatomical and RAPD-PCR analyses.

ABSTRACT

Bombacoideae and Malvoideae are two subfamilies belong to family Malvaceae sensu lato (s. l.). These two subfamilies had long been problematic at the taxonomical level. Moreover, some genera have not been adequately sampled for sufficiently variable molecular markers. In this work, we selected three species ( , and ) that are related to Bombacoideae and Malvoideae. They were mainly selected because they are commonly present in Egypt, and exhibit high medicinal values and contain Silk fiber in their which is valuable in fabric industry. In this study, we compared these three from the taxonomical point of view as an initial step to determine their characteristic features, and investigated their genomic DNA banding patterns using randomly amplified bands (RAPD- PCR). This was done not only to determine the phylogenetic relationships but also to find new molecular markers that will help in their specific characterization.

Key words: Bombacoideae- Malvoideae- Egypt -RAPD PCR- , Ceiba pentandra, Ceiba speciosa.

Introduction was the reason to combine Malvaceae s.s and into Malvaceae s.l. [2,10,19] to form The large family Malvaceae sensu lato the clade Malvatheca [7]. Baum et al [8] observed comprises of approximately 4300 species and 250 that within clade Malvatheca, there are two major genera [11. The recent circumscription recognized lineages: Bombacoideae and Malvoideae. this family on the basis of a number of Bombacoideae includes 14 genera and 160 species morphological and molecular data which divided it from the family Bombacaceae [10]. Bombacaceae is into four traditional families: , predominately exhibit tropical [33,32] that are Bombacaceae, and Malvaceae sensu distributed in America, Africa, Asia and Australia stricto (s.s.) [1,2,7,8,10,54,43,41]. In addition, [38]. Malvoideae includes the traditional Malvaceae traditional circumscription dealt with Malvaceae (Mallows or Eumalvoideae) that comprises 78genera (s.s.) as a very homogenous and cladistically and 1700 species [8]. The relationships between the monophyletic group [13,14,15,47,48,51,52], whereas two major lineages: Bombacoideae and Malvoideae it considered the other three families as paraphyletic still largely unresolved and problematic at the [33,32]. Malvaceae s.l. exhibits stellate hairs and taxonomical level [1,2,7,8,11,41]. Thus, the objective palmately veined with inflorescence structures of this study was to determine the interrelationships consisting of bicolor units, valvate sepals, mucilage among the three species from the two subfamilies of cavities within cortex & pith and cyclopropanoid oil Malvacea s.l. Bombax ceiba Linn. (Bombacoideae), [10,11,35]. On the other hand, Bombacaceae Ceiba pentandra (L.) Gaertn. and Ceiba speciosa exhibits habit, have polyandrous with (A.St. HiL.) Ravenna (Malvoideae) they are unilocular anthers and smooth pollen [14,48,28]. commonly present in Egypt and are famous by their Although, there were a close relationships between medicinal, economical and nutritional values, were Malvaceae s.s. and Bombacaceae, most classification selected for this purpose. systems, [3,11], and references [26,49] have Here, the taxonomical characterization will maintained them as separate families. Recent depend on detailed morphological, anatomical phylogenetic analysis using atpB ,rbcL, and ndhF studies while the molecular characterization will genes from chloroplast and ITS gene from nucleus depend on DNA banding patterns resulted from their Coressponding Author Noha S. Khalifa, Botany Dept., Faculty of Women for Arts, Science, and Education Ain Shams University. E-mail: [email protected] 416 Adv. Environ. Biol., 7(2): 416-426, 2013 genomic DNA using RAPD- PCR primers. This HCl, 0.05 EDTA, 1.25% SDS, pH 8) then incubated study is mainly done to determine the (1) at 60 ºC for 3 h. The temperature of homogenate is phylogenitic relationships & their evolution (2) to brought to room temperature and then 250 µl of cold find new molecular markers that can be attributed in 6M ammonium acetate was added, shacked their specific characterization. vigorously and incubated for 30 min at 4ºC. Samples were centrifuged in (Sigma centrifuge, 2K15, USA) Material and Method at 5000 rpm for 30 minutes at 4 ºC to collect the precipitated proteins and tissue. 600 µl of the Plant sample collection: supernatant were recovered into new collection micro tubes containing 360 µl of iso-propanol mix Bombax ceiba Linn. silk-cotton tree, Ceiba thoroughly and allow the DNA to precipitate pentandra (L.) Gaertn., kapok, Ceiba speciosa (A. overnight at -20ºC. Centrifuge for 15 minutes at 5000 ST. Hill.) Ravenna Silk Floss tree were collected of rpm to pellet DNA. The pellet was washed twice in year 2010 from different regions through out Cairo 500 µl of 70% ethanol and centrifuged for 15 city during the vegetative and flowering times minutes at 5000 rpm. The pellet was resuspended in January-March, May-October, July-November of the least amount of ddH2O and left overnight to each plant respectively . dissolve at 4°C. The purity and concentration of extracted genomic DNA were checked by Morphological and anatomical studies: spectrophotometer (PG Instrument Limited, T90+, and U.K.). DNA concentrations were calculated The morphological study of different plant using the formula [DNA = optical density (OD260) × organs was described either directly from the tree at dilution factor × constant (50 μg/ ml)]. DNA samples its location or from fresh specimens. Averages of were diluted to 50-100 ng/µl in sterile distilled water dimension and lengths of leaflet's and and stored at -20 °C. The integrity and concentration petiolule for at least 20 specimens were measured in of the DNA was confirmed by running samples on cm. For anatomical study, cross microtome sections 1% (w/v) agarose gel electrophoresis for 45 min at of 10-20 µm were cut from stems, petioles and 80 V. DNA bands were visualized using UV leaves, stained with safranin - light green mixture transilluminator (Spectroline, TX 312,USA) after following the method described by Johanson [31] staining with ethidium bromide (Bioshop, Canada and photographed under light microscope. The Inc.) morphological and anatomical descriptions were conducted according to Willis (1973) and Metcalfe Optimization of RADP PCR analysis: and Chalk [39], respectively. A total of 20 ten mer RAPD primers were used Morphological and anatomical data analysis: in this study, of which 5 combinations were finally selected based on polymorphism, quality and Morphological and anatomical characters were reproducibility of the amplifications (Table 1). Due given the numerical code 0 or 1 according to their to the high sensitivity of the PCR- RAPD technology absence or presence, respectively. The similarity to changes in experimental parameters, primers were matrix was determined using NTSYS-PC software initially screened against the three plants (bulked (version 2.02, Rohlf, 1998). Phylogenetic leaves DNA). Optimization includes the adjustments dendrogram was generated from the similarity matrix of magnesium, template DNA concentrations, pH following the Unweighted Pair Group Method using values, length of the denaturation stage and primer Arithmetic Averages (UPGMA; Sneath and Sokal;, annealing temperature. The highest performance of 1973). primers is observed at 34 °C. Reactions were performed by volume of 20 μL in 5X Green GoTaq DNA extraction: Reaction Buffer pH 8.5 containing blue and yellow dyes, 7.5 mM magnesium. (M3008 promega, USA), DNA was extracted from fresh young leaves and 2.5 mM dNTPs (Promega, USA), 25 pmol of each was pooled from 20 different plants. Due to the high primers (Metabion International AG, Germany), 3 ng viscosity of the material, commercially available DNA and 0.3 U of Taq polymerase and ddH2O. PCR kits such as DNazol, Trizole and plant DNA thermal cycler (Technie TC-4000, UK) was extraction kit failed to extract high quality DNA for programmed as follows, 94 °C for 5 min, followed PCR analysis. Thus, a new method was adopted here by 35 cycles of 40 sec at 94 °C, 40 sec at 34 °C, and for genomic DNA extraction. Fresh leaves were 3 min at 72 °C. The PCR tubes were kept at 72 °C ground in liquid N2 and 0.3 g from each sample was for 10 min as a final extension step and then stored at homogenized in 0.5 ml extraction buffer (0.1M Tris- 4 °C until analyzed using gel electrophoresis.

417 Adv. Environ. Biol., 7(2): 416-426, 2013

Table 1: List of RAPD primers combination that gave reproducible and polymorphic results with the three plants used in this study. # Primer pair Sequence '5-'3 GC content% A r34 GTCACCGGA 60% r1302.1 GGAAATCGTG 50% B r34 GTCACCGGA 60% OPA1 CAGGCCCTTC 70% C r34 GTCACCGGA 60% OPP2 TCGGCACGAC 70% D r34 GTCACCGGA 60% OPM13 GGTGGTCAAC 60% E r11 CCAAGCAGT 50% r1302.1 GGAAATCGTG 50%

Analysis of DNA banding patterns: and needs further revision mainly due to the lack of more detailed systematic and phylogenetic studies. RAPD bands were separated electrophoretically This had greatly deprived these plants from the using 1% agarose gel in 1x TAE buffer, stained with discovery of an adequate molecular marker that can ethedium bromide and photographed on a UV- distinguish them apart. transilluminator using a digital camera (12Mp,Sony). DNA from each plant was amplified with the same Analysis of morphological and anatomical data: pair of primers 3 times and the banding patterns were compared to make sure that the result is reproducible. All the morphological and anatomical characters All visible and unambiguous bands were scored and of the three species studied are showed in (Tables recorded using PyElph 1.4 software system for gel 2&3), (Figs.1,2,3), and were used for cluster analysis image analysis and phylogenetics (Pavel and Vasile, Fig.(4). 2012). Bands appear in only one plant scored as Table (2) shows that Bombax ceiba is unique, those present in two scored as polymorphic, morphologically distinct from Ceiba pentanda and and bands present in three plants are common bands. Ceiba speciosa in thorn shape & color, trunk Polymorphic primers were used to verify the linkage developed with butteresses, tree branching type, of marker within each trait. large pulvinus, petiole length (14.5cm), leaflet petiolule length (2.6cm), leaflet blade length Data analysis for PCR-RAPD: (11cm)& width (4.8cm) where Bombax ceiba has the highest values, the number, shape and apex of the According to [29], RAPD primers behave as leaflet and different flowering seasons. These results dominant markers and can be used with a bistate were concomitant with previous studies [12,8]. It is (present-absent) type of scoring. Digital images of known that morphology is detrimental in ethidium bromide stained gels were used to score the evolution and the three species under study are data for RAPD analysis. Each DNA fragment coming from one common ancestor [32]. Yet, amplified by a given primer was treated as a unit Bombax ceiba flowers conserved the ancestral character and each fragment was scored as present condition which is represented by synapomorphies (1) or absent (0) for each of the primer pair characters such as large flower with fleshy cup- combination used and this was recorded in a binary shaped calyx, polyandrous stamens with unilocular data matrix. The resulting similarity coefficients anthers that possess stakled filaments forming five were used to evaluate the relationships among the bundles united at base with calyx & corolla and three plants with cluster analysis using an produce tremendous amount of pollen which favors unweighted pair group method with arithmetic pollination via mammals like bats [20]. averages (UPGMA). This information was used for On the other hand, the creamy-white flowers of plotting the dendrogram using NTSYSPC program Ceiba pentandra are small and have only five version 2.0.1.5 (Applied biostatistics Inc., USA). It is stamens arranged in spherical fasciculate noteworthy to mention that the band intensity was inflorescences, they are pollinated by a wide range of not considered and the fragments with identical flying & non flying mammals such as bees, wasps mobility were scored as identical ones. The and [24]. Ceiba speciosa flowers are molecular size of the amplification products was relatively, pink colored and have staminal tube with determined from 1Kb ladder (SM0323, Fermentas sessile anthers, Table (2) . Many Ceiba species have Inc., USA). staminodial appendages that are associated with a filament tube and limit access to the nectar, they are Results and Discussion present in pollinated species (eg. Ceiba speciosa) but absent or reduced in most bat- The three species studied have high economical, pollinated species [23,22]. The bat, bird and insect medicinal, and nutritional values, but yet they are pollination were coincident with extreme undroecial problematic at the taxonomical level. Thus, the modification [6]. current classification of those plants is contradictory

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[30] recoded that the Malvatheca clade have bat– of species with polyandrous flowers of stalked pollinated flowers with elongated sessile monothecate stamens have evolved in Bombacoideae monothecate stamens represent the ancestral and Malvoideae may indicate species radiation in condition in the clade. He added to, the large number connection with switch from bat to insect pollination.

Table 2: Morphological characters and character state of the three Species studied: 1- Bombax ceiba Linn. 2 - Ceiba pentandra (L.) Geartn. & 3 - Ceiba speciosa (A. St. Hill.) Ravenna. Character State Species 1 2 3 whole plant Habit Trees 1 1 1 1 1 1 Texture Thorny 1 1 1 Trunk shape Butresses 1 1 0 Bottle shape 0 0 1 Trunk colour Green 0 0 1 Gray 1 0 0 Brown 0 1 0 Thorn shape Pointed conical 1 0 0 Corase conical 0 1 1 Thorn color Green 0 0 1 Grayish 1 0 0 Brown 0 1 0 Branching pattern World 1 0 0 Densely crowded 0 1 0 Horizontal 0 0 1 leaves Stipules Present 1 1 1 Deciduous 1 1 1 Pulvinus Present 1 1 1 Pulvinus size Small 0 1 1 Large 1 0 0 Petiole color Green 1 0 1 Red 0 1 0 Length of leaf petiole (cm) 4.5 Cm 0 0 1 13.5 Cm 0 1 0 14.5 Cm 1 0 0 Length of leaflet petiolule (cm) 0.6 Cm 0 1 1 2.6 Cm 1 0 0 Leave blade type Palmately compound 1 1 1 Leaflet number Five 1 0 0 Nine 0 1 1 Leaflet blade shape Broad obovate 1 0 0 Elibitic lanceolate 0 1 0 Lanceolate 0 0 1 leaflet blade length (cm) 4 Cm 0 0 1 10 Cm 1 1 0 11 Cm 1 0 0 leaflet blade width (cm) 2.3 Cm 0 0 1 4 Cm 0 1 0 4.8 Cm 1 0 0 Leaflet margin Entire 1 1 0 Serrate 0 0 1 Leaflet apex Acute 0 0 1 Acuminate 1 1 0 Flowers type Solitary cymose 1 0 1 Fascicled cymose 0 1 0

Table 1: continue Flower size Small 0 1 0 Large 1 0 1 Flower color White 0 1 0 Pink 0 0 1 Red 1 0 0 Flowering period January - Mars 1 0 0 May - October 0 1 0 July - November 0 0 1

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Flower sex Bisexual 1 1 1 Flower symmetry Actinomirphic 1 1 1 Calyx number 5- Gamosepalus 1 1 1 Calyx shape Cup-shaped 1 0 0 Tubular 0 0 1 Campanulate 0 1 1 Calyx coherent United with corolla &stamens 1 0 0 Corolla shape Obovate 1 0 0 Oblong-spathulate 0 1 0 Oblong with wavy margins 0 0 1 Corolla coherent Joined at base with stamens 0 1 1 Stamens structure & number Numerous forming fivebundles united at base 1 0 0 Five stamens united at base in a staminal tube 0 1 0 Five staminal tube with sessile anther 0 0 1 Anther cells one - celled 1 0 0 Two - celled 0 1 1 Filament attachment anther Basy fixed or dorsy 1 1 1 Anther facing Extrose 1 1 1 Ovary position Semi - inferior 0 1 0 Superior 1 0 1 Carpels coherent Syncarbus 1 1 1 Number of Carples Five - carpels 1 1 1 type & dehiacence & Loculicidal dehiscent 1 1 1 type Oily endospermic 1 1 1 Seed germination Epigeal 1 1 1 Seeds size & color Small & blak 1 0 1 Large & black 0 1 0 Fibers color White 1 0 0 Brown 0 1 0

At the anatomical level, Ceiba pentanda and five festoon shape in Bombax ceiba (Fig. 1B,C). The Ceiba speciosa are similar to each other and differed leaf mid rib contains open vascular bundles in Ceiba from Bombax ceiba Table (3). In Ceiba pentanda pentanda and Ceiba speciosa (Figs. 2 C, 3C) while it and Ceiba speciosa stem, the pericycle is marked by is closed in Bombax ceiba (Fig. 1C). strands of fibers situated at the outer periphery of the Metcalfe and Chalk [39] recoded that the more phloem groups, phloem strands usually stratified into complex vasculature within the petiole of fibrous and non fibrous zones where primary Bombacaceae seems to be associated with arboreal medullary rays present between them in triangular habit. It had been reporteded that Bombax ceiba shape with inward apex whereas the phloem in exhibits low density hardwood and could be utilized Bombax ceiba forms a continuous ring. In addition, in making matchwood [40,18]. On the other hand, clear annual rings only were observed within the Ceiba pentandra wood is characterized by thicker xylem of Ceiba pentanda (Figs. 1A, 2A, 3A). The fiber wall, less parenchymatous tissue, presence of main vascular cylinder within the petiole forms four anomalous structure in phloem and longitudinal gum closed vascular bundles in Ceiba pentanda and Ceiba canals [17]. These results were in agreement with out speciosa (Figs. 2 B,C & 3 B,C) whereas it forms data

Table 3: Anatomical characters and character state of the three Species studied: 1- Bombax ceiba Linn. 2 - Ceiba pentandra (L.) Geartn. & 3 - Ceiba speciosa (A. St. Hill.) Ravenna. Characters State Species Stem 1 2 3 Cross section Wavy 0 1 0 Terete 1 0 0 Cuticle layer Thin 1 0 0 Thick 0 1 1 Epidermal layer One layer 0 1 1 Tow layers 1 0 0 Number of cortex layers 10 - 12 layers 0 1 1 18 - 20 layers 1 0 0 Cortical Cell type Parenchyma 1 1 1 Collenchyma 1 1 1 Mucilage cavities 1 1 1 Tanniniferous cells 1 1 1 Pericycle Dissected cylinder of fibrous 1 0 1 Stratified zones as triangular shape 0 1 0 Phloem Dissected cylinder 1 0 0 Stratified into fibrous and non fibrous zones 0 1 1

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Phloem medullary rays Horizontal 1 0 0 Triangular with inword apex 0 1 1 Xylem Nearly closed cylinder 1 0 1 Closed cylinder interrupted by lignified medullary rays 0 1 0 Intervascular rays Narrow bands, one cell wide 1 0 1 Narrow bands up to three cells wide 0 1 0 Pith Solid 1 1 1 Crystal type Solitary & Cluster 1 1 1 Petiole Cross section Lobed 1 1 1 Cuticle layer Thick 1 1 1 Epidermal layer One 0 1 1 Cortical cell type Parenchyma 1 1 1 Collenchyma 1 1 1 Tanniniferous cells 1 1 1 Pericycle Strands of fibers 1 1 1 Crystal type Solitary & Cluster 1 1 1 Number of vascular bundles Four 0 1 1 Six 1 0 0 Pith Solid 1 1 1 Leaf Cuticle layer Thick 1 1 1 Epidermal cells Two cells 1 1 1 Mesophyll type Dorsiventral 1 1 1 Mid rib vascular bundle Closed vascular cylinder 1 0 0 Open vascular cylinder 0 1 1 Cortical layers Mucilage cavities 1 1 1 Tanniniferous cells 1 1 1 Crystal type Solitary Cluster 1 1 1

The obtained dendrogram from morphological pentandra and Ceiba speciosa (Fig.5, Table4). All and anatomical analysis divided the three species into primers combination revealed characteristic two groups at the taxonomic distance of 0.69. Group fragments that were detected in one plant but not I had Bombax ceiba (1) at the single level, and group present in the others (Fig 5. Table 5). Interestingly, II had Ceiba pentandra (2) and Ceiba speciosa (3) in all combinations were able to show high genetic one cluster at the taxonomic distance of 0.46 (Fig. 4). diversity among the plants tested. The five combinations of primers generated 69 reproducible Phylogenetic analysis: and scorable amplification products across all plants, out of which 61 (~ 88.41%) fragments were Beside the morphological and anatomical polymorphic among which 41 (67.21%) unique and information, molecular DNA polymorphism can 20 (32.78%) non unique bands were detected. The greatly contribute to the use of genetic diversity percentage of polymorphism was high and reached through the descriptive information they provide on 100% in D, 92.8% in A, 90.9% in C, 83.3% in E and the structure of gene pools and identify unique 76.47% in B. The proportions of common bands molecular markers of genetically polymorphic traits were low (10.14%). [9,37,53]. The ability of this method to distinguish According to the RAPD- PCR data for DNA between taxa helps in botanical quality analysis [34]. banding patterns the dendrogram showed that The technique was found useful to study markers on Bombax ceiba was separated at the genetic all the linkage groups, genotypes distinctness, distance/Coefficient of 1.45 while the other two genetic distances and classification of accessions into species were separated at 1.34 (Fig. 6). Bombax specific groups. ceiba is also separated at the taxonomical distance Our analysis revealed that out of 20 117, while Ceiba pentandra and Ceiba speciosa combinations of RAPD primers pair, 5 combinations were separated at level 88 when both taxonomic and gave reproducible and polymorphic results when phylogenetic dendrogram were combined Fig (7). used with DNA extracted from Bombax ceiba, Ceiba

Table 4: Number of total bands, monomorphic (common), polymorphic bands and percentage of polymorphism revealed by the 10mer random primers used to amplify the DNA of 1-Bombax ceiba, 2 -Ceiba pentadra and3- Ceiba speciosa using RAPD-PCR technique. polymorphic bands Primers Total No. Of bands monomorphic bands unique Non unique polymorphism% A 14 1 8 5 92.86 B 17 4 8 5 76.47 C 11 1 6 4 90.9 D 15 0 13 2 100

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E 12 2 6 4 83.3 Total 69 8 41 20

Table 5: The distribution and molecular weight marker of unique bands (markers) revealed by RAPD among the examined samples of1- B. ceiba , 2- C. Pentandra and3- C. speciosa. Primer set A B C D E M. Wt. (bp) 2000 C. speciosa B. ceiba C. pentandra B. ceiba 1300 B. ceiba C. speciosa C. speciosa 1250 B. ceiba 1200 C. pentandra C. speciosa 1100 C. speciosa B. ceiba C. pentandra 1000 B. ceiba C. pentandra 950 B. ceiba C. pentandra 930 C. speciosa 900 C. speciosa 870 C. speciosa B. ceiba 830 C. speciosa C. speciosa B. ceiba 810 C. pentandra C. speciosa 800 B. ceiba 790 B. ceiba 750 C. speciosa B. ceiba 730 C. pentandra B. ceiba 700 C. speciosa 690 B. ceiba 670 B. ceiba B. ceiba C. pentandra 650 B. ceiba B. ceiba 610 B. ceiba 500 C. pentandra

Fig. 1: Light microscopy photographs of trans-sections of plant organs of Bombax ceiba. (A) Stem, 100x. (B) Petiole; 17x, (C) 100x. (D) Leaf, 32

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Fig. 2: Light microscopy photographs of trans-sections of plant organs of Ceiba pentandra. (A) Stem, 100x. (B) Petiole; 17x, (C) 100x. (D) Leaf, 32

Fig. 3: Light microscopy photographs of trans-sections of plant organs of Ceiba speciosa. (A) Stem, 100x. (B) Petiole; 17x, (C) 100x. (D) Leaf, 32x.

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Fig. 4: The DNA banding patterns which were resulted from the combinations of two primer pairs. 1 is B. Ceiba, 2 is C. pentandra, 3 is C. speciosa. Letters from A-D represent the primer combination as shown in Table 1.

Fig. 5: A dendrogram showing the morphology and anatomy relationships among the three species 1- Bombax ceiba Linn. 2- Ceiba pentandra (L.) Geartn. &3-Ceiba speciosa (A. St. Hill.) Ravenna.

Fig. 6: A dendrogram showing the genetic relationships among the three species 1- Bombax ceiba Linn. 2: Ceiba pentandra (L.) Geartn. & 3-Ceiba speciosa (A. St. Hill.) Ravenna.

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Fig. 7: Combined dendrogram showing the morphology & anatomy and genetic relationships among the three species 1 - Bombax ceiba Linn. 2- Ceiba pentandra (L.) Geartn. & 3 - Ceiba speciosa (A. St. Hill.) Ravenna.

Conclusion: the Linnean society, 141: 399-436 (Available online: Abstract full text: full text (PDF). This study revealed that: 5. Angiosperm Phylogeny Website- Valvales. 1- Taxonomical comparison is concomitant Missouri Batanical Garden Press. with RAPD-PCR result in detecting phylogenetic 6. Balthazar, M., von W.S. Alverson, J. interrelation ships. Schönenberger, D.A. Baum, 2004. Comparative 2- One can compare as less as three species floral development and androecium structure in and still can get the same result. Malvoideae (Malvaceae s.l.). Int J Plant Sci., 3- Finding new primers that are helpful in 165: 445-473. distinguishing between the three species used in this 7. Baum, D.A., W.S. Alverson, R. Nyffeler, 1998a. study at the molecular level. A by any other name: and 4- The revision of this classification was nomenclature of the core . Harvard Pap consistent with the previous results Bot, 3: 315-330. 5- There is clear evolution in the three species 8. Baum, D.A., S.D. Smith, A. Yen, W.S. particularly via the androcium modification, the Alverson, R. Nyffeler, B.A. Whitlock, R.L. flower size and pollination methods with Ceiba Oldham, 2004. Phylogenetic relationships of pentandra being more likely the intermediate link Malvatheca (Bombacoideae and Malvoideae; between Bombax ceiba and Ceiba speciosa. Malvaceae sensulato) as inferred from plastid DNA sequences Amer J Bot., 91: 1862-1870. References 9. Brown-Guedira, G.L., J.A. Thompson, R.L. Nelson, M.L. Warburton, 2001. Evaluation of 1. Alverson, W.S., K.G. Karol, D.A. Baum, M.W. genetic diversity of soybean introductions and Chase, S.M. Swensen, R. McCourt, K.J. Sytsma, North American ancestors using RAPD and SSR 1998. Circumscription of the Malvales and markers. Crop Sci., 40: 815-823. relationships to other Rosidae: evidence from 10. Bayer, C., M.F. Fay, A.Y. De Bruijn, V. rbcL sequence data. Amer J Bot., 85: 876-887. Savolainen, C.M. Morton, K. Kuitzki, W.S. 2. Alverson, W.S., B.A. Whitlock, R. Nyffeler, C. Alverson, M.W. Chase, 1999. Support for an Bayer, D.A. Baum, 1999. Phylogeny of the Core expanded family concept of Malvaceae within Malvales: Evidence from ndhF sequence data. recircumscribed order Malvales: a combined Amer J Bot., 86: 1476-1486. analysis of plastid atpB and rbcL DNA 3. Angiosperm Phylogeny Groub, I (1998), II sequences. Bot. J. Linn. Soc., 129: 267-303. (2003) 11. Bayer, C., K. Kubitzki, 2003. Malvaceae. In K. 4. Angiosperm phylogeny Group, 2003. An update Kubitzki and C. Bayer [eds.]. The families and of the Angiosperm phylogeny Group genera of vascular plants. 5: 225-311 Springer, classification for the orders and families of Berlin, Germany. flowering plants: APG IL. Botamical Journal of

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