Bangladesh J. Plant Taxon. 26(2): 205–218, 2019 (December) © 2019 Bangladesh Association of Plant Taxonomists DNA BARCODING AND MORPHO-ANATOMICAL CHARACTERS OF TWO FORMS OF CONVOLVULUS ARVENSIS L. (CONVOLVULACEAE) GROWN IN ASIR REGION, SAUDI ARABIA MAHMOUD MOUSTAFA*, SAAD ALAMRI1, ALI SHATI, MOHMED AL-KAHTANI, SULAIMAN ALRUMMAN AND ABDELRAHEEM TAWFEK2 Department of Biology, College of Science, King Khalid University, 9004, Abha, Kingdom of Saudi Arabia (KSA) Keywords: Convolvulus arvensis L.; rbcL; matK; ITS. Abstract DNA barcoding and morpho-anatomical characters of two forms of Convolvulus arvensis L. growing in Asir region, Saudi Arabia were studied. We applied three molecular markers, ITS, rbcL and matK for phylogenetic reconstruction in combination with 24 morpho-anatomical characters to provide proposal for the recognition of its population under two forms. Plant samples were collected from ten various populations of C. arvensis supposed to be of two different forms, and finally these have been found to be of two new forms (small leaf) and form (large leaf) for the flora of Saudi Arabia. TWINSPAN results showed that there was negative group with twenty one objects including stem length, stem thickness, seed shapes, petiole length, leaf apex, leaf length, petal circumference, leaf width, capsule length, capsule width, stamen length, anther width, anther length, carpel length, venations type, pollen grains shape, T.S of stem, root and leaf, stigma shape and anther base surface and positive group including hairs, stomata and anther shape. Dendrogram upon morpho-anatomical characters resolved two main clusters, one cluster of form (large leaf) and the other of form (small leaf). Amplified gene region for the form (small leaf)-rbcL showed 100% identity with C. lineatus; form (small leaf)-matK and form (large leaf)-matK had 99% identity with Calystegia sepium (L.) R. Br. Form of (small leaf)–ITS had 100% identity with that of C. arvensis; form (large leaf)-rbcL had 100% identity with those of C. arvensis and C. lineatus. Form (large leaf)–ITS had 99% identity with that of C. arvensis. The results of molecular phylogenetic analyses based on certain morphological and rbcL data support two main clusters within C. arvensis which is consistent with two forms, form (small leaf) and form (large leaf). Introduction Convolvulus arvensis L. is one of the most common weeds all over the world found mostly in about 54 countries distributed in 32 different economic crops (Holm et al., 1997). It was named as a field-bind weed due to its extensive deep root system together with long-term seed bank that were considered as key features to the noxious weed status (Gianoli, 2001). C. arvensis plants are native to Europe and grows extensively in Mediterranean climates, temperate and tropical regions and in many other climatic zones (Lyons, 1998). Field bindweed was considered as a perennial vine of the glorious family Convolvulaceae which spreads by rhizome or/and seed and its prostrate weak -stem, often twine forming tangled dense mats (Wiese and Phillips, 1976; Gleason and *Corresponding author. Email: mfmostfa@kku.edu.sa. 1Prince Sultan Bin Abdulaziz Center For Environmental and Tourism Research and Studies-King Khalid University (KSA). 2Department of Botany, Faculty of Science, South Valley University, Qena, Egypt. mailto:mfmostfa@kku.edu.sa. 206 MOUSTAFA et al. Cronquist, 1963). Its underground rhizomes can extend between 5 cm and 2.6 m and deeply penetrate the lands and its climbing stems can reach up to 1.5 meter. Till now, no infraspecific category for C. arvensis has been reported in Saudi Arabia (Collenette, 1985; Migahid, 1978 and 1996) while one variety has been registered in the North America, namely C. arvensis var. obtusifolium Choisy (Robinson and Fernald 1908). Gray (1970) also opined that there were two forms of field bindweed, one had leaves with broad basal lobes and on wide cordate base named as form cardifolius Lasch. and the other one had acute ear-shaped lobes on the base of the leaves and an oblong, linear, or lanceolate blades named as form auriculatus Descr. In Europe more than sixty varieties of C. arvensis have been characterized and the description used to sort out among them have been attributed to environmental conditions (Kogan, 1986). In USA, it has been found that there were many intermediate characteristics to the identified varieties of C. arvensis, however, researchers were discouraging to apply specific name to them (Brown, 1946). The Kingdom of Saudi Arabia (KSA) contains one of the most diverse floras among the surrounding countries due to its various climatic regime such as Mediterranean, semiarid, and arid climates and cold climate in high altitude areas of 2700 m above sea level. These environmental conditions in KSA might affect C. arvensis populations to be splitted in to two or more forms, and based on this ground we contemplate to examine whether two or more forms of C. arvensis are growing in KSA. In the past, the study of plant taxonomy was based to a large extent on the morphological characters of the floral parts. Recently, an application of molecular techniques in taxonomy is being increasingly used to interpret the phylogenetic relationship among plant taxa. Molecular techniques have emerged as an accurate additional tool in providing authentic and unambiguous identification of specific taxa and to study boundaries among specific species (Kaukas and Rollinson 1997). DNA sequence data have notably helped with a high level of accuracy in reconstruction of the phylogeny of different groups of organisms presenting totally new understandings on their taxonomy and phylogeny (Friesen et al., 2006; Asmussen et al., 2006; Lefébure et al. 2006). Also, many data previously obtained from different sources other than genetic materials are being neglected. Hence, this study was carried out to evaluate the morphological, anatomical and molecular variations in C. arvensis of Asir region, KSA, in order to investigate the existence of any infra specific taxon and understand the phylogenetic relationships within this species based on DNA sequence data obtained from matK, rbcL and ITS regions. Materials and Methods Morpho-anatomical character states Vegetative characters including stem length, stem thickness, petiole length, leaf apex, leaf length, petal circumference, leaf width, capsule length, capsule width, stamen length, anther width, anther length, carpel length, venations type, pollen grains shape, stigma shape, anther base surface, hairs, stomata, anther shape, seed shape, transverse section in stem, in root and in leaves, were studied either with the naked eye or under a stereo microscope (Leica, 10446322, 2X WD). Five populations for each of the two forms viz. form (small leaf) and form (large leaf) were selected and characteristics of five individuals from each of this population were investigated. The measurement was considered as an averaged plus/minus maximum and minimum for various quantitative parameters. According to Stevens (1991) all described characters were quantitatively and qualitatively expressed by denoting a coding value, to prevent misrepresentation of the possible variation among the investigated plants. DNA BARCODING AND MORPHO -ANATOMICAL CHARACTERS 207 Statistical analysis The multivariate statistical analysis for morpho-anatomical characters was carried out using Community Analysis Package (CAP) Statistics program, version 5. Cluster analysis for the morpho-anatomical characters including (Euclidean Ward's (Nath et al., 2014; Ward, 1963), TWINSPAN (Two Way Indicator Species Analysis, Manhas et al., 2009) and Jaccard‘s Similarity Coefficient were performed. DNA barcoding Genomic DNA from form (small leaf) and form (large leaf) from healthy fresh leaf were extracted using a Qiagen DNAeasy Plant Mini kit. matK forward (5'-ACCCAGTCC ATCTGGAAATCTTGGTTC-3'), and matK reverse (5'–CGTACAGT ACTTTTGTGTTTACGA G-3') and rbcL forward (5'–ATGTCACCACAAACAGAGACTAAAGC-3'), rbcL reverse (5'–GT AAAATCAAGTCCACCRCG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (Costion et al., 2011;Yuan et al., 2015) regions were amplified by applying Polymerase Chain Reaction (PCR) technique. 50 μL reaction solutions having, 1×PCR reaction buffer, 0.2 mM deoxynucleotide triphosphate (dNTPs), 2.5 mM MgCl2, 0.2 μM each primer, 1 U of Taq DNA polymerase and 0.75–1.5 μL genomic DNA of two examined extracts for amplifications process. PCR program was adjusted as followings: 3 min at 94°C for initial denaturation, 35 cycles of 30 Sat 94°C for denaturation, 30 S at 48°C/ 57°C for annealing, 1 min at 72°C for primer extension, and a final extension for 10 min at 72°C. All products gained from PCR and stained with ethidium bromide were visualized on 1% agarose gel and a UV table. By using 1kb DNA ladder the amplification of the primer had been confirmed and estimated. The amplified bands were introduced for sequencing by using Applied Biosystems, 3500 genetic analyser. Resulted sequence from primer was compared with each other by using Bio edit version 7.1.11 and Clustal W multiple sequence alignment to investigate molecular phylogenetic analysis (Tamura et al., 2011; Grimm et al., 2006). Obtained sequences from the matK, rbcL and ITS regions for the two taxa were compared by other relevant sequences using BLAST (Basic Local Alignment Search Tool). A split is a bipartition in a species set, that divides all dataset of species into two groups: a functional out-group and a functional in-group (Huson et al., 2010). Results and Discussion Morpho-anatomical features The list of morphological and anatomical characters that were investigated together with their character states is shown in the Figs 1.1 - 1.5 and Table 1. The resulted dendrogram based on these characters was resolved in two main discrete clusters of form (small leaf) and form (large leaf) for the ten populations of which were separated at linkage distance of 52.9. While five populations of form (small leaf) were further resolved in to two sub-clusters at 1.2 (Fig. 2). The Jaccard‘s pairwise similarity coefficient values for ten populations of C. arvensis ranged from 0.0833 to 1.0, whereas those among the populations of form (small leaf) varied from 0.96 to 1.00 and of form (large leaf) didn’t vary, rather remained always highest (1.00) (Table 2). These values between the populations of two forms (small leaf) and form (large leaf) ranged from 0.0833 to 0.125. Whereas, this coefficient was 0.0833 between P2 and P4 of forms (small leaf) and P1-P5 of forms (large leaf) and it was 0.125 between P1, P3 and P5 of forms (small leaf) and P1-P5 of forms (large leaf, Table 2). TWINSPAN output for the classification of ten populations revealed that there was negative group with twenty one characters including stem length, stem thickness, seed shapes, petiole length, leaf apex, leaf length, petal circumference, leaf width, capsule length, capsule width, 208 MOUSTAFA et al. stamen length, anther width, anther length, carpel length, venations type, pollen grains shape, transverse section (TS) in stem, in root, in leaf, stigma shape and anther base surface. Also it showed that there was positive group of three characters comprising hairs, stomata and anther shape. Fig.1.1. Filed images of habit (A1), leaf (A2) and venations (A3) of C. arvensis form small leaf and habit (B1), leaf (B2) and venations (B3) of C. arvensis form large leaf. Fig.1.2. Hairs (A1), stomata (A2) and leaf apex (A3) of C. arvensis L. form small leaf and hairs (B1), stomata (B2) and leaf apex (B3) of C. arvensis form large leaf. Fig.1.3. Anther (A1); anther base surface (A2); Bifid stigma (A3) and stigma rod (A4) of C. arvensis form small leaf and anther (B1); anther base surface (B2); bifid stigma (B3) and stigma rod (B4) of C. arvensis form large leaf. Fig. 1.4. Pollen grains (A1, A2 and A3) of C. arvensis L. form small leaf and (B1, B2 and B3) of C. arvensis form large leaf. DNA BARCODING AND MORPHO -ANATOMICAL CHARACTERS 209 Fig.1.5. Transverse section (T.S.) of stem (A1); root (A2 and A3); leaf (A4) of C. arvensis L. form small leaf and T.S. of stem (B1); root (B2 and B3); and leaf (B4) of C. arvensis form large leaf. As the morpho-anatomical characters of C. arvensis from Saudi Arabia showed distinct variation between the two forms, namely form (small leaf) and form (large leaf). Therefore, these variations can be used to clearly distinguish between some closely related subspecies, varieties, and forms. Fig. 2. Dendrogram based on morpho-anatomical characters from ten populations of C. arvensis form (small leaf (sl)) and form (large leaf (ll)) of C. arvensis. 210 MOUSTAFA et al. Table 1. Morphological and anatomical characters for C. arvensis form (small leaf) and C. arvensis form (large leaf). Characters Characters states of C. arvensis form (small leaf) from five populations Characters states of C. arvensis form (large leaf) from five populations Stem length 99.45 cm (Max 105 cm ± Min 90 cm) almost prostrate 224.57 cm (Max 270 cm ± Min 190 cm) climbing Stem thickness 0.97 ml (Max 1.1ml ± Min 0.88 ml) 1.21 ml (Max 1. 28 ml ± Min 1.15 ml) Seed shapes Short (small)-3 seeds - beared shaped Long-3 seeds – beared shaped Petiole length Shortened, 6.7 ml (Max 7.1 ml ± Min 6.00 ml) (Fig 1.1.A2) Flattened, 25.72 ml (Max 26.0 ml ± Min 25.0 ml) (Fig 1.1.B2) Leaf apex Acuminate on the upper side (Fig.1.2. A3) Mucronate on the upper side (Fig.1.2. B3) Leaf length 46.32 ml (Max 47.7 ml ± Min 45 ml) (Fig 1.1. A2) 111.47. ml (Max 115 ml ± Min 110 ml) (Fig 1.1.B2) Petal circ. 64.31 ml (Max 65.49 ml ± Min 60.92 ml) (Fig 1.1. A1) 94.33 ml(Max 97.03 ml ± Min 90.34 ml) (Fig 1.1. B1) Leaf width 15.08 ml (Max 15.3 ml ± Min 14.8 ml) (Fig 1.1. A2) 40.40 ml(Max 41.2 ml ± Min 39.98 ml) (Fig 1.1.B2) Capsule length 5.58 ml (Max 5.9 ml ± Min 5.1 ml) 9 ml (Max 9.7 ml ± Min 9.4 ml) Capsule width 3.26 ml (Max 3.4 ml ± Min 3.10 ml) 5.26 ml (Max 5.50 ml ± Min 5.10 ml) Stamen length 9.25 ml (Max 9.4 ml ± Min 9.1 ml (Fig.1.3. A2) 11.49 ml (Max 13.0 ml ± Min 10.1 ml (Fig.1.3. B2) Anther width 0.09 ml (Max 0.10 ml ± Min 0.08 ml) (Fig.1.3. A1) 0.12 ml (Max 0.14 ml ± Min 0.12 ml) (Fig.1.3. B1) Anther length 2.16 ml (Max 2.22 ml ± Min 2.12 ml) (Fig.1.3. A1) 2.28 ml (Max 2.41 ml ± Min 2.23 ml) (Fig.1.3. B1) Carpel length Style: 11.4 ml (Max 11.5 ml ± Min 11.1 ml; (Fig.1.3. A3); stigma: 3.16 ml (Max 3.23 ml ± Min 3.10 ml) (Fig.1.3. A4) Style: 12.24 ml (Max 12.4 ml ± Min 12.1 ml); (Fig.1.3. B3); stigma: 3.26 ml (Max 3.33 ml ± Min 3.20 ml) (Fig.1.3. B4) Venations From 6 to 7; nearly even pinnate (Fig.1.1. A3) From 7 to 8; alternate (Fig.1.1. B3) Pollen grains In cluster with three grooves (Fig. 1.4. A1, 2 and 3) Solitary or in triplex with prominent three grooves (Fig. 1.4. B1, 2 and 3) Anther shape Sagitate (Fig. 1.3. A1) Sagitate (Fig. 1.3. B1) Stigma shape Bifid (Fig. 1.3. A3) Bifid (Fig. 1.3. B3) Anther base surface Cylindrical (Fig. 1.3. A2) Cylindrical (Fig. 1.3. B2) Hairs Many types mainly compounds (Fig.1.2. A1) Many types mainly compounds (Fig.1.2. B1) Stomata Kidneys shapes of stomata (Fig.1.2. A2) Kidneys shapes of stomata (Fig.1.2. B2) T.s in Stem Fig.1.5. A1 Fig.1.5. B1 T.S. in Root Fig.1.5. A2 and A3 Fig.1.5. B2 and B3 T.S. in leaf Fig.1.5. A4 Fig.1.5. B4 DNA BARCODING AND MORPHO -ANATOMICAL CHARACTERS 211 Table 2. Jaccard‘s similarity coefficient among 24 characters of two forms (small leaf (sl)) and (large leaf (ll)) of C. arvensis. P1. form (sl) P2. form (sl) P3. form (sl) P4. form (sl) P5. form (sl) P1. form (sl) 1.0000 P2. form (sl) 0.9583 1.0000 P3. form (sl) 1.0000 0.9583 1.0000 P4. form (sl) 0.9583 1.0000 0.9583 1.0000 P5. form (sl) 1.0000 0.9583 1.0000 0.9583 1.000 P1. form (ll) 0.125 0.08333 0.125 0.08333 0.125 P2. form (ll) 0.125 0.08333 0.125 0.08333 0.125 P3. form (ll) 0.125 0.08333 0.125 0.08333 0.125 P4. form (ll) 0.125 0.08333 0.125 0.08333 0.125 P5. form (ll) 0.125 0.08333 0.125 0.08333 0.125 Table 2. (Contd.) Jaccard‘s similarity coefficient among 24 characters of two forms (small leaf (sl)) and form (large leaf (ll)) of C. arvensis. P. form (ll) P2. form (ll) P. form (ll) P4. form (ll) P5. form (ll) P1. form (sl) P2. form (sl) P3. form (sl) P4. form (sl) P5. form (sl) P1. form (ll) 1.0000 P2. form (ll) 1.0000 1.0000 P3. form (ll) 1.0000 1.0000 1.0000 P4. form (ll) 1.0000 1.0000 1.0000 1.0000 P5. form (ll) 1.0000 1.0000 1.0000 1.0000 1.0000 This conclusion is consistent with Sa`ad (1967) who distinguished between the morphologically very similar species C. valentinusis and C. supinus on the light of the minor variations in their leaf shapes and the presence or absence of hairs on both sides of their leaves . DNA sequence analysis The resulted sequence length of the matK, rbcL and ITS regions for the two forms of C. arvensis were in between 577-862 bp for form (large leaf) -rbcL and form (small leaf)-matK respectively (Table 3). The GC content ranged between 33.874 for form (small leaf)-matK to 56.086 for form (small leaf)-ITS. Form (small leaf)-rbcL showed 100% identity with C. lineatus (KT178135.1) and C. arvensis (KM360729.1), (100%). Form (small leaf)-matK and form (large leaf)-matK had 99% identity with C. arvensis (KT176616.1), C. arvensis (MG946999.1), Calystegia sepium (JN894556.1) and Calystegia sepium (FJ395438.1). Form (small leaf)-ITS had 100% identity with C. arvensis (AY560274.1), C. arvensis (AY558826.1), C. arvensis 212 MOUSTAFA et al. (KC528905.1), C. arvensis (KJ021876.1) and C. arvensis (JQ062475.1). Form (large leaf)-rbcL had 100% identity with C. arvensis (KT178135.1), C. lineatus (MF158799.1) and C. arvensis (KM360729.1). Form (large leaf)-ITS had 99% with C. arvensis (AY560274.1), C. arvensis (AY558826.1), C. arvensis (KC528905.1), C. arvensis (KJ021876.1), and C. arvensis (JQ062475.1). Table 3. Read Length, GC Content and BlastN Report of rbcL, matK and ITS regions of form (small leaf) and form (large leaf). Form name Read Length GC Content BlastN Report (Description –Accession no. and Identities Pct.(%) form (small leaf)- rbcL 582 45.70446735395189 C. arvensis (KT178135.1), (99 %) C. arvensis (MG946887.1), (99 %) C. lineatus (KT178135.1), (100 %) C. arvensis (KM360729.1), (100 %) C. arvensis (AY100993.1), (99 %) form (small leaf)- matK 862 33.874709976798144 C. arvensis (KT176616.1), (99 %) C. arvensis (MG946999.1), (99 %) Calystegia sepium (JN894556.1), (99 %) C. cneorum (HQ384565.1), (98 %) Calystegia sepium (FJ395438.1), (99 %) form (small leaf)- ITS 690 56.086956521739125 C. arvensis (AY560274.1), (100 %) C. arvensis (AY558826.1), (100 %) C. arvensis (KC528905.1), (100 %) C. arvensis (KJ021876.1), (100 %) C. arvensis (JQ062475.1), (100 %) form (large leaf)- rbcL 577 45.407279029462735 C. arvensis (KT178135.1), (100 %) C. lineatus (MF158799.1), (100 %) C. arvensis (MG946887.1), (99 %) C. arvensis (KM360729.1), (100 %) C. arvensis (AY100993.1), (99 %) form (large leaf)- matK 856 33.8785046728972 C. arvensis (KT176616.1), (99 %) C. arvensis (MG946999.1), (99 %) Calystegia sepium (JN894556.1), (99 %) C. cneorum (HQ384565.1), (98 %) Calystegia sepium (FJ395438.1), (99 %) form (large leaf)- ITS 680 55.88235294117647 C. arvensis (AY560274.1), (99 %) C. arvensis (AY558826.1), (99 %) C. arvensis (KC528905.1), (99 %) C. arvensis (KJ021876.1), (99 %) C. arvensis (JQ062475.1), (99 %) The output of identity matrix sequences resulted from rbcL, matK and ITS are shown (Figure 3 and Table 4).The sequence identity matrix revealed that the highest identity matrix ranged between 100% among form (large leaf)-ITS, form (small leaf)-rbcL and form (small leaf)-rbcL, DNA BARCODING AND MORPHO -ANATOMICAL CHARACTERS 213 followed by 99.3% among form (large leaf)-matK and form (small leaf)-matK. The next highest sequence identity matrix 97.8% was found among form (large leaf)-ITS, form (small leaf)-rbcL and form (small leaf)-ITS. The lowest sequence identity matrix 26.3% was recorded among form (small leaf)-ITS and form (large leaf)-rbcL (Table 4). Fig. 3. Sequence alignment for rbcL, matK and ITS region forms (small leaf (sl)) and form (large leaf (ll)) of C. arvensis. 214 MOUSTAFA et al. Table 4. Sequence identity matrix for rbcL, matK and ITS genes region of form (small leaf(sl)) and form (large leaf (ll)) of C. arvensis. form (sl) - rbcL form (ll) -rbcL Form (sl) -matK form (ll) -matK Form (sl) -ITS form (ll) -ITS form (sl) -rbcL ID 0.263 0.352 0.355 0.978 1.000 form (ll) -rbcL 0.263 ID 0.283 0.285 0.258 0.263 form (sl) -matK 0.352 0.283 ID 0.993 0.354 0.352 form (ll) -matK 0.355 0.285 0.993 ID 0.356 0.355 form (sl) -ITS 0.978 0.258 0.354 0.356 ID 0.978 form (ll) -ITS 1.000 0.263 0.352 0.355 0.978 ID The maximum likelihood (ML) analysis was based on rbcL, matK and ITS regions of two forms of C. arvensis (Figure 4). The ML tree was resolved in to two clusters of the two forms. Cluster 1 formed at the branch length of 0.8 was consisted of form (small leaf)-rbcL + ITS and form (large leaf)-ITS. Cluster 2 at branch length of 0.3 was consisted of the clade of form (large leaf)-matK and form (small leaf)-matK. The branch length of the out-group form (large leaf)-rbcL was 0.8. Fig. 4. ML tree generated through maximum likelihood analysis of rbcL, matK and ITS sequence data of two forms (small leaf (sl)) and form (large leaf (ll)) of C. arvensis. The nucleotide composition resulted from rbcL, matK and ITS regions of two forms of C. arvensis are shown in (Table 5). The average of all the three nucleotide sequences had a total of 724 positions, in the final data set, for Pos #1 (241), for Pos #2 and for Pos #3 (240). It revealed that form (small leaf)-rbcL had the highest Guanine (G) content (28.7%) and lowest Adenine (A) (20.6%), while form (large leaf)-rbcL had the highest Thymine (T) and Adenine contents of 27.4% and 27.2% respectively and lowest Cytosine (C) content of 22.0. Form (small leaf)-matK and form (large leaf)-matK had the highest Thymine (37.7%) and lowest Guanine (15.9%). Form (small leaf)-ITS and form (large leaf)-ITS had high amount of Guanine (287%) and low amount of Adenine (20.6%). The degree of bias showed avraiation depends upon the codon composition. At the first codon position for form (small leaf)-rbcL, the usage of G was 33.3%, and those of the other bases were 25.4% (C), 19.3% (A) and 22.0% (T). At the second codon position, the content DNA BARCODING AND MORPHO -ANATOMICAL CHARACTERS 215 of T was 23.00% and those of the other bases were 28.6% (C), 22.00% (A) and 26.4% (G). At the third codon position, the base usage was T (26.00%), C (27.6%), A (20.4%) and G (26.2%) (Shrivastava et al., 2013). The detailed numbers of codon composition for form (large leaf)-rbcL, form (small leaf)-matK, form (large leaf)-matK, form (small leaf)-ITS, form (large leaf)- ITS are shown in Table 5. Table 5. Nucleotide composition of rbcL, matK and ITS regions of form (small leaf (sl)) and form (large leaf (ll)) of C. arvensis. Source T(U) C A G Total T-1 C-1 A-1 G-1 Pos #1 form (sl)-rbcL 23.5 27.2 20.6 28.7 680.0 22 25.4 19.3 33.3 228.0 form (ll)-rbcL 27.4 22.0 27.2 23.4 577.0 34 20.9 27.2 18.3 191.0 form (sl)-matK 37.7 18.0 28.4 15.9 862.0 37 15.3 29.6 18.1 287.0 form (ll)-matK 37.7 18.0 28.4 15.9 856.0 37 15.1 29.8 17.9 285.0 form(sl)-ITS 23.2 27.2 20.7 28.8 690.0 22 25.0 19.8 33.2 232.0 form (ll)- ITS 23.5 27.2 20.6 28.7 680.0 22 25.4 19.3 33.3 228.0 Avg. 29.6 22.9 24.6 22.9 724.2 29 20.7 24.5 25.3 241.8 Table 5. (Contd.) Nucleotide composition of rbcL, matK and ITS regions of form (small leaf (sl)) and form (large leaf (ll)). Source T-2 C-2 A-2 G-2 Pos #2 T-3 C-3 A-3 G-3 Pos#3 form (sl)-rbcL 23 28.6 22.0 26.4 227.0 26 27.6 20.4 26.2 225.0 form (ll)- rbcL 21 18.7 25.9 34.2 193.0 27 26.4 28.5 17.6 193.0 form (sl)-matK 36 17.7 27.1 18.8 288.0 40 20.9 28.6 10.8 287.0 form (ll)- matK 36 17.8 26.9 18.9 286.0 40 21.1 28.4 10.9 285.0 form (sl)- ITS 23 28.7 22.2 26.5 230.0 25 28.1 20.2 26.8 228.0 form (ll)- ITS 23 28.6 22.0 26.4 227.0 26 27.6 20.4 26.2 225.0 Avg. 28 23.0 24.5 24.5 241.8 31 24.9 24.7 19.1 240.5 Phylogenetic relationships based on molecular and morphological properties have been considered in many taxa (Bernardi and Crane, 2005; Ward et al., 2005). Also, combinations between DNA sequencing and morphological characters including leaf anatomy and macromorphology had been conducted to investigate the generic and subgeneric relations (Soh and Parnell, 2011). Interestingly in this work, we found concordant results from the analyses of molecular data and morphological and anatomical characters. We found variations in morphological and anatomical characters accompanied with nucleotide sequences of two forms of C. arvensis. Such variations may be due to the differences in the GC percentage, nucleotide length, nucleotide bases differences, and number of gaps in each nucleotide region (Singh et al., 2016). These differences in each form of C. arvensis can be mainly ascribed to the varying numbers of repeat, alignment gaps, deletions, copy number sand base 216 MOUSTAFA et al. substitutions and/or additions. Base additions and substitutions are featured by very high C content that might form pure poly C structures (Shrivastava et al., 2013). Therefore, all of these factors individually or altogether will lead to a consequence of mutations in the lineage of C. arvensis forms according the prevailing environmental conditions. Variation in the amount of GC content and other nucleotides A, T and C in the first, second and third position is same to that of some other plant groups, for example, Triticeae tribe (Bieniek et al., 2015) and Pisum sativum varieties (Moustafa et al., 2019). Comparison the obtained sequences from rbcL, matK and ITS regions with the sequences in the GenBank gave various similarity percentage with other species of convolvulus plants. For example, form (small leaf)-rbcL and form (large leaf)-rbcL gave a 100% similarity with C. lineatus and C. arvensis. There was no 100% similarity was obtained from form (small leaf)- matK, and form (large leaf)-matK, though form (small leaf)-ITS gave 100% similarity with C. arvensis. Also, the molecular phylogenetic analyses showed that form (large leaf) –rbcL clustered alone than other nucleotide sequences generated from matK and ITS regions. Therefore, rbcL can be applied to address the problems of plant phylogeny in lower taxa, even at the level of form, since it could separate form (large leaf) from form (small leaf) and in concomitant with morpho- anatomical characters of these two forms. However Baldwin et al. (1995), found that ITS could reconstruct phylogenetic relationships precisely among plant species. Li et al. (2011) found that ITS effectively resolved the problems of species by sorting out 6,286 samples from 1,757 of seed plant species. An application of ITS was also questioned. The sequencing and amplification rate of ITS primer was relatively low (i.e. 71.00% and 86.20% respectively) which might be as a result from some second-level structures on it (Huang et al., 2015; DeSalle, 2007; Waugh, 2007). Additionally, Xing et al. (2018) found that rbcL and matK were not appropriated for characterization the plant for tropical forest at the species level. 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