Comparative study of three species of Malvatheca (Bombacoideae and Malvoideae (Malvaceae sensu lato) using morphological, anatomical and RAPD-PCR analyses.
The large family Malvaceae sensu lato comprises of approximately 4300 species and 250 genera [11. The recent circumscription recognized this family on the basis of a number of morphological and molecular data which divided it into four traditional families: Tiliaceae, Bombacaceae, Sterculiaceae and Malvaceae sensu stricto (s.s.) [1,2,7,8,10,54,43,41]. In addition, traditional circumscription dealt with Malvaceae (s.s.) as a very homogenous and cladistically monophyletic group [13,14,15,47,48,51,52], whereas it considered the other three families as paraphyletic [33,32]. Malvaceae s.l. exhibits stellate hairs and palmately veined leaves with inflorescence structures consisting of bicolor units, valvate sepals, mucilage cavities within cortex & pith and cyclopropanoid oil seed [10,11,35]. On the other hand, Bombacaceae exhibits tree habit, have polyandrous flowers with unilocular anthers and smooth pollen [14,48,28]. Although, there were a close relationships between Malvaceae s.s. and Bombacaceae, most classification systems, [3,11], and references [26,49] have maintained them as separate families. Recent phylogenetic analysis using atpB, rbcL, and ndhF genes from chloroplast and ITS gene from nucleus was the reason to combine Malvaceae s.s and Bombacaceae into Malvaceae s.l. [2,10,19] to form the clade Malvatheca . Baum et al  observed that within clade Malvatheca, there are two major lineages: Bombacoideae and Malvoideae. Bombacoideae includes 14 genera and 160 species from the family Bombacaceae . Bombacaceae is predominately exhibit tropical trees [33,32] that are distributed in America, Africa, Asia and Australia . Malvoideae includes the traditional Malvaceae (Mallows or Eumalvoideae) that comprises 78genera and 1700 species . The relationships between the two major lineages: Bombacoideae and Malvoideae still largely unresolved and problematic at the taxonomical level [1,2,7,8,11,41]. Thus, the objective of this study was to determine the interrelationships among the three species from the two subfamilies of Malvacea s.l. Bombax ceiba Linn. (Bombacoideae), Ceiba pentandra (L.) Gaertn. and Ceiba speciosa (A.St. HiL.) Ravenna (Malvoideae) they are commonly present in Egypt and are famous by their medicinal, economical and nutritional values, were selected for this purpose.
Here, the taxonomical characterization will depend on detailed morphological, anatomical studies while the molecular characterization will depend on DNA banding patterns resulted from their genomic DNA using RAPD-PCR primers. This study is mainly done to determine the (1) phylogenitic relationships & their evolution (2) to find new molecular markers that can be attributed in their specific characterization.
Material and Method
Plant sample collection:
Bombax ceiba Linn. silk-cotton tree, Ceiba pentandra (L.) Gaertn., kapok, Ceiba speciosa (A. ST. Hill.) Ravenna Silk Floss tree were collected of year 2010 from different regions through out Cairo city during the vegetative and flowering times January-March, May-October, July-November of each plant respectively.
Morphological and anatomical studies:
The morphological study of different plant organs was described either directly from the tree at its location or from fresh specimens. Averages of dimension and lengths of leaflet's petiole and petiolule for at least 20 specimens were measured in cm. For anatomical study, cross microtome sections of 10-20 [micro]m were cut from stems, petioles and leaves, stained with safranin--light green mixture following the method described by Johanson  and photographed under light microscope. The morphological and anatomical descriptions were conducted according to Willis (1973) and Metcalfe and Chalk , respectively.
Morphological and anatomical data analysis:
Morphological and anatomical characters were given the numerical code 0 or 1 according to their absence or presence, respectively. The similarity matrix was determined using NTSYS-PC software (version 2.02, Rohlf, 1998). Phylogenetic dendrogram was generated from the similarity matrix following the Unweighted Pair Group Method using Arithmetic Averages (UPGMA; Sneath and Sokal;, 1973).
DNA was extracted from fresh young leaves and was pooled from 20 different plants. Due to the high viscosity of the leaf material, commercially available kits such as DNazol, Trizole and plant DNA extraction kit failed to extract high quality DNA for PCR analysis. Thus, a new method was adopted here for genomic DNA extraction. Fresh leaves were ground in liquid N2 and 0.3 g from each sample was homogenized in 0.5 ml extraction buffer (0.1M Tris-HCl, 0.05 EDTA, 1.25% SDS, pH 8) then incubated at 60[degrees]C for 3 h. The temperature of homogenate is brought to room temperature and then 250 [micro]l of cold 6M ammonium acetate was added, shacked vigorously and incubated for 30 min at 4[degrees]C. Samples were centrifuged in (Sigma centrifuge, 2K15, USA) at 5000 rpm for 30 minutes at 4[degrees]C to collect the precipitated proteins and plant tissue. 600 [micro]l of the supernatant were recovered into new collection micro tubes containing 360 [micro]l of iso-propanol mix thoroughly and allow the DNA to precipitate overnight at -20[degrees]C. Centrifuge for 15 minutes at 5000 rpm to pellet DNA. The pellet was washed twice in 500 [micro]l of 70% ethanol and centrifuged for 15 minutes at 5000 rpm. The pellet was resuspended in the least amount of dd[H.sub.2]O and left overnight to dissolve at 4[degrees]C. The purity and concentration of extracted genomic DNA were checked by spectrophotometer (PG Instrument Limited, [T90.sup.+], and U.K.). DNA concentrations were calculated using the formula [DNA = optical density (OD260) x dilution factor x constant (50 [micro]g/ ml)]. DNA samples were diluted to 50-100 ng/[micro]l in sterile distilled water and stored at -20[degrees]C. The integrity and concentration of the DNA was confirmed by running samples on 1% (w/v) agarose gel electrophoresis for 45 min at 80 V. DNA bands were visualized using UV transilluminator (Spectroline, TX 312,USA) after staining with ethidium bromide (Bioshop, Canada Inc.)
Optimization of RADP PCR analysis:
A total of 20 ten mer RAPD primers were used in this study, of which 5 combinations were finally selected based on polymorphism, quality and reproducibility of the amplifications (Table 1). Due to the high sensitivity of the PCR-RAPD technology to changes in experimental parameters, primers were initially screened against the three plants (bulked leaves DNA). Optimization includes the adjustments of magnesium, template DNA concentrations, pH values, length of the denaturation stage and primer annealing temperature. The highest performance of primers is observed at 34[degrees]C. Reactions were performed by volume of 20 [micro]L in 5X Green GoTaq Reaction Buffer pH 8.5 containing blue and yellow dyes, 7.5 mM magnesium. (M3008 promega, USA), 2.5 mM dNTPs (Promega, USA), 25 pmol of each primers (Metabion International AG, Germany), 3 ng DNA and 0.3 U of Taq polymerase and ddH2O. PCR thermal cycler (Technie TC-4000, UK) was programmed as follows, 94[degrees]C for 5 min, followed by 35 cycles of 40 sec at 94[degrees]C, 40 sec at 34[degrees]C, and 3 min at 72[degrees]C. The PCR tubes were kept at 72[degrees]C for 10 min as a final extension step and then stored at 4[degrees]C until analyzed using gel electrophoresis.
Analysis of DNA banding patterns:
RAPD bands were separated electrophoretically using 1% agarose gel in 1x TAE buffer, stained with ethedium bromide and photographed on a UV-transilluminator using a digital camera (12Mp,Sony). DNA from each plant was amplified with the same pair of primers 3 times and the banding patterns were compared to make sure that the result is reproducible. All visible and unambiguous bands were scored and recorded using PyElph 1.4 software system for gel image analysis and phylogenetics (Pavel and Vasile, 2012). Bands appear in only one plant scored as unique, those present in two scored as polymorphic, and bands present in three plants are common bands. Polymorphic primers were used to verify the linkage of marker within each trait.
Data analysis for PCR-RAPD:
According to , RAPD primers behave as dominant markers and can be used with a bistate (present-absent) type of scoring. Digital images of ethidium bromide stained gels were used to score the data for RAPD analysis. Each DNA fragment amplified by a given primer was treated as a unit character and each fragment was scored as present (1) or absent (0) for each of the primer pair combination used and this was recorded in a binary data matrix. The resulting similarity coefficients were used to evaluate the relationships among the three plants with cluster analysis using an unweighted pair group method with arithmetic averages (UPGMA). This information was used for plotting the dendrogram using NTSYSPC program version 220.127.116.11 (Applied biostatistics Inc., USA). It is noteworthy to mention that the band intensity was not considered and the fragments with identical mobility were scored as identical ones. The molecular size of the amplification products was determined from 1Kb ladder (SM0323, Fermentas Inc., USA).
Results and Discussion
The three species studied have high economical, medicinal, and nutritional values, but yet they are problematic at the taxonomical level. Thus, the current classification of those plants is contradictory and needs further revision mainly due to the lack of more detailed systematic and phylogenetic studies. This had greatly deprived these plants from the discovery of an adequate molecular marker that can distinguish them apart.
Analysis of morphological and anatomical data:
All the morphological and anatomical characters of the three species studied are showed in (Tables 2&3), (Figs.1,2,3), and were used for cluster analysis Fig.(4).
Table (2) shows that Bombax ceiba is morphologically distinct from Ceiba pentanda and Ceiba speciosa in thorn shape & color, trunk developed with butteresses, tree branching type, large pulvinus, petiole length (14.5cm), leaflet petiolule length (2.6cm), leaflet blade length (11cm)& width (4.8cm) where Bombax ceiba has the highest values, the number, shape and apex of the leaflet and different flowering seasons. These results were concomitant with previous studies [12,8]. It is known that flower morphology is detrimental in evolution and the three species under study are coming from one common ancestor . Yet, Bombax ceiba flowers conserved the ancestral condition which is represented by synapomorphies characters such as large flower with fleshy cup-shaped calyx, polyandrous stamens with unilocular anthers that possess stakled filaments forming five bundles united at base with calyx & corolla and produce tremendous amount of pollen which favors pollination via mammals like bats .
On the other hand, the creamy-white flowers of Ceiba pentandra are small and have only five stamens arranged in spherical fasciculate inflorescences, they are pollinated by a wide range of flying & non flying mammals such as bees, wasps and hummingbirds . Ceiba speciosa flowers are relatively, pink colored and have staminal tube with sessile anthers, Table (2). Many Ceiba species have staminodial appendages that are associated with a filament tube and limit access to the nectar, they are present in butterfly pollinated species (eg. Ceiba speciosa) but absent or reduced in most bat-pollinated species [23,22]. The bat, bird and insect pollination were coincident with extreme undroecial modification .
 recoded that the Malvatheca clade have bat-pollinated flowers with elongated sessile monothecate stamens represent the ancestral condition in the clade. He added to, the large number of species with polyandrous flowers of stalked monothecate stamens have evolved in Bombacoideae and Malvoideae may indicate species radiation in connection with switch from bat to insect pollination.
At the anatomical level, Ceiba pentanda and Ceiba speciosa are similar to each other and differed from Bombax ceiba Table (3). In Ceiba pentanda and Ceiba speciosa stem, the pericycle is marked by strands of fibers situated at the outer periphery of the phloem groups, phloem strands usually stratified into fibrous and non fibrous zones where primary medullary rays present between them in triangular shape with inward apex whereas the phloem in Bombax ceiba forms a continuous ring. In addition, clear annual rings only were observed within the xylem of Ceiba pentanda (Figs. 1A, 2A, 3A). The main vascular cylinder within the petiole forms four closed vascular bundles in Ceiba pentanda and Ceiba speciosa (Figs. 2 B,C & 3 B,C) whereas it forms five festoon shape in Bombax ceiba (Fig. 1B,C). The leaf mid rib contains open vascular bundles in Ceiba pentanda and Ceiba speciosa (Figs. 2 C, 3C) while it is closed in Bombax ceiba (Fig. 1C).
Metcalfe and Chalk  recoded that the more complex vasculature within the petiole of Bombacaceae seems to be associated with arboreal habit. It had been reporteded that Bombax ceiba exhibits low density hardwood and could be utilized in making matchwood [40,18]. On the other hand, Ceiba pentandra wood is characterized by thicker fiber wall, less parenchymatous tissue, presence of anomalous structure in phloem and longitudinal gum canals . These results were in agreement without data
The obtained dendrogram from morphological and anatomical analysis divided the three species into two groups at the taxonomic distance of 0.69. Group I had Bombax ceiba (1) at the single level, and group II had Ceiba pentandra (2) and Ceiba speciosa (3) in one cluster at the taxonomic distance of 0.46 (Fig. 4).
Beside the morphological and anatomical information, molecular DNA polymorphism can greatly contribute to the use of genetic diversity through the descriptive information they provide on the structure of gene pools and identify unique molecular markers of genetically polymorphic traits [9,37,53]. The ability of this method to distinguish between taxa helps in botanical quality analysis . The technique was found useful to study markers on all the linkage groups, genotypes distinctness, genetic distances and classification of accessions into specific groups.
Our analysis revealed that out of 20 combinations of RAPD primers pair, 5 combinations gave reproducible and polymorphic results when used with DNA extracted from Bombax ceiba, Ceiba pentandra and Ceiba speciosa (Fig.5, Table 4). All primers combination revealed characteristic fragments that were detected in one plant but not present in the others (Fig 5. Table 5). Interestingly, all combinations were able to show high genetic diversity among the plants tested. The five combinations of primers generated 69 reproducible and scorable amplification products across all plants, out of which 61 (~ 88.41%) fragments were polymorphic among which 41 (67.21%) unique and 20 (32.78%) non unique bands were detected. The percentage of polymorphism was high and reached 100% in D, 92.8% in A, 90.9% in C, 83.3% in E and 76.47% in B. The proportions of common bands were low (10.14%).
According to the RAPD-PCR data for DNA banding patterns the dendrogram showed that Bombax ceiba was separated at the genetic distance/Coefficient of 1.45 while the other two species were separated at 1.34 (Fig. 6). Bombax ceiba is also separated at the taxonomical distance 117, while Ceiba pentandra and Ceiba speciosa were separated at level 88 when both taxonomic and phylogenetic dendrogram were combined Fig (7).
This study revealed that:
1--Taxonomical comparison is concomitant with RAPD-PCR result in detecting phylogenetic interrelation ships.
2--One can compare as less as three species and still can get the same result.
3--Finding new primers that are helpful in distinguishing between the three species used in this study at the molecular level.
4--The revision of this classification was consistent with the previous results
5--There is clear evolution in the three species particularly via the androcium modification, the flower size and pollination methods with Ceiba pentandra being more likely the intermediate link between Bombax ceiba and Ceiba speciosa.
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(1) Wafaa M. Said, (2) Nahla O.M. Ehsan, (3) Noha S. Khalifa
(1,2) Botany Dept., Faculty of Women for Arts, Science, and Education Ain Shams University.
(3) Botany Dept., Faculty of Science, Ain Shams University
Noha S. Khalifa, Botany Dept., Faculty of Women for Arts, Science, and Education Ain Shams University.
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% 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 Deciduous 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 4.5 Cm 0 0 1 petiole (cm) 13.5 Cm 0 1 0 14.5 Cm 1 0 0 Length of leaflet 0.6 Cm 0 1 1 petiolule (cm) 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 Broad obovate 1 0 0 shape Elibitic lanceolate 0 1 0 Lanceolate 0 0 1 leaflet blade 4 Cm 0 0 1 length (cm) 10 Cm 1 1 0 11 Cm 1 0 0 leaflet blade 2.3 Cm 0 0 1 width (cm) 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 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 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 Numerous forming fivebundles 1 0 0 & number united at base Five stamens united at base 0 1 0 in a staminal tube Five staminal tube with 0 0 1 sessile anther Anther cells one-celled 1 0 0 Two-celled 0 1 1 Filament Basy fixed or dorsy 1 1 1 attachment anther Anther facing ovary 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 Fruit type & Capsule & Loculicidal 1 1 1 dehiacence dehiscent Seeds 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 Table 3: Anatomical characters and character state of the three Species studied: 1--Bombax ceiba Linn. 2--Ceibapentandra (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 10-12 layers 0 1 1 cortex layers 18-20 layers 1 0 0 Cortical Cell Parenchyma 1 1 1 type Collenchyma 1 1 1 Mucilage cavities 1 1 1 Tanniniferous cells 1 1 1 Pericycle Dissected cylinder 1 0 1 of fibrous Stratified zones as 0 1 0 triangular shape Phloem Dissected cylinder 1 0 0 Stratified into 0 1 1 fibrous and non fibrous zones Phloem medullary Horizontal 1 0 0 rays Triangular with 1 1 inword apex Xylem Nearly closed 1 1 cylinder Closed cylinder 1 interrupted by lignified medullary rays Intervascular rays Narrow bands, 1 1 one cell wide Narrow bands up 1 to three cells wide 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 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 Four 1 1 bundles Six 1 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 Closed vascular 1 bundle cylinder Open vascular 1 1 cylinder Cortical layers Mucilage cavities 1 1 1 Tanniniferous cells 1 1 1 Crystal type Solitary Cluster 1 1 1 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 and 3--Ceiba speciosa using RAPD-PCR technique. Primers Total monomorphic polymorphic bands polymorphism% No. Of bands bands unique Non unique 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 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 of 1--B. ceiba, 2--C. Pentandra and 3--C. speciosa. Primer set A B C M. Wt. (bp) 2000 C. speciosa B. ceiba C. pentandra 1300 B. ceiba 1250 1200 C. pentandra 1100 C. speciosa 1000 B. ceiba C. pentandra 950 B. ceiba C. pentandra 930 900 C. speciosa 870 C. speciosa B. ceiba 830 C. speciosa 810 C. pentandra 800 B. ceiba 790 750 C. speciosa 730 C. pentandra 700 C. speciosa 690 670 B. ceiba B. ceiba 650 610 B. ceiba 500 Primer set D E M. Wt. (bp) 2000 B. ceiba 1300 C. speciosa C. speciosa 1250 B. ceiba 1200 C. speciosa 1100 B. ceiba C. pentandra 1000 950 930 C. speciosa 900 870 830 C. speciosa B. ceiba 810 C. speciosa 800 790 B. ceiba 750 B. ceiba 730 B. ceiba 700 690 B. ceiba 670 C. pentandra 650 B. ceiba B. ceiba 610 500 C. pentandra
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|Title Annotation:||random amplified polymorphic DNA-polymerase chain reaction; ORIGINAL ARTICLE|
|Author:||Said, Wafaa M.; Ehsan, Nahla O.M.; Khalifa, Noha S.|
|Publication:||Advances in Environmental Biology|
|Date:||Feb 1, 2013|
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