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Comparative study of three species of Malvatheca (Bombacoideae and Malvoideae (Malvaceae sensu lato) using morphological, anatomical and RAPD-PCR analyses.

Introduction

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 [7]. Baum et al [8] observed that within clade Malvatheca, there are two major lineages: Bombacoideae and Malvoideae. Bombacoideae includes 14 genera and 160 species from the family Bombacaceae [10]. Bombacaceae is predominately exhibit tropical trees [33,32] that are distributed in America, Africa, Asia and Australia [38]. Malvoideae includes the traditional Malvaceae (Mallows or Eumalvoideae) that comprises 78genera and 1700 species [8]. 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 [31] and photographed under light microscope. The morphological and anatomical descriptions were conducted according to Willis (1973) and Metcalfe and Chalk [39], 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 extraction:

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 [29], 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 2.0.1.5 (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 [32]. 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 [20].

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 [24]. 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 [6].

[30] 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 [39] 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 [17]. 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).

Phylogenetic analysis:

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 [34]. 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).

Conclusion:

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

Coressponding Author

Noha S. Khalifa, Botany Dept., Faculty of Women for Arts, Science, and Education Ain Shams University.

E-mail: dr.wafaamorsy@hotmail.com

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
Article Type:Report
Geographic Code:7EGYP
Date:Feb 1, 2013
Words:5180
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