Abstract: Background and Objective: Petiole anatomy of 30 species of Asteraceae belonging to 24 genera was undertaken to investigate variations in vascular bundle arrangement at the basal and distal of the petiole including the petiole outline to determine their taxonomic significance. Materials and Methods: Methods used in this study were petiole sectioning using a sliding microtome, staining, mounting and observation under a light microscope. Results: Our findings showed that all species studied have mainly three major vascular bundles and minor ones which formed either an open separate arc or open continuous/united arc with rib traces. Five different types of vascular bundle arrangements were described based on the number, size, arrangement, presence or absence of accessory and/or interspersed vascular bundles and 8 petiole outlines. Among the vascular bundle types, type-1 occurred in 19 species and 12 genera, type-2 in 8 species and 7 genera, type-3 in 3 species and 3 genera, type-4 in 12 species and 11 genera type-5 in 1 specie and 1 genus. The type-5 vascular bundle is restricted to V. conferta. The petiole outline type-1 occurred in 9 species, type-2 in 5 species, type-3 in 16 species, type-4 in 5 species, type-5 in 11 species, type-6 in 3 species, type-7 and 8 in 1 specie each. Twenty three of the species had a leaf-like wing at either distal or basal end of the petiole. Conclusion: These characters are fairly different and diagnostic especially the presence and number of accessory and interspersed vascular bundles among the taxa especially at the genus and species level.
INTRODUCTION
Petiolar anatomical features are one of the key parameters used in the identification and classification of many plant families and have been used in differentiating plants of different species, genera and families1-5, but are imperfectly studied2. In recent times, there is increased interest and research on the use of petiole anatomy as a complementary tool in plant taxonomy and significant progress has been made in using this line of evidence in plant classification6-8. Different authors have also considered the arrangement of vascular bundles in different portions of the petiole to actually have taxonomic relevance2,3,9. Metcalfe and Chalk2-3 emphasized that the distal end (the portion immediately below the lamina) has considerable taxonomic features compared to other parts of the petiole.
Asteraceae is considered one of the largest plant family with approximately 950-1500 genera and 25,000 species in different habitats10,11. Eighty-four genera and 273 species occur in West Africa12. They exhibit mainly herbaceous habits, although arboreous and voluble herbaceous habits also occur13,14. Because of the variety of habits, the family presents various anatomical structures and in some cases, ecological specialization may occur3,15 but the anatomy of the petiole being sparsely or not affected2. Among the members of this family, considerable works have been done on the secretory structures being restricted in distribution and as an important diagnosis character3,16 by occupying distinct positions in different organs of the plant17,18. The anatomical features such as epidermal and lamina characteristics of some members of this family have been described to have various types of glandular or covering trichomes, papillae on the abaxial epidermis, having anomocytic, anisocytic and rarely heliocytic stomata, presence of hydathodes, presence of hypoderm, vascular bundles with parenchymatic sheath composed by large cells1,19. Also, this family presents species of great importance in the fields of nutrition, cosmetics and pharmacy due to the production of essential oils20,21.
The present study aims to complement the above treatments by examining a relatively large number of species from Nigeria. In total, the petiole of 30 Asteraceae species was studied in order to provide additional data for the identification of the species.
MATERIALS AND METHODS
Source of the sample used: Representative specimens deposited in the University of Port Harcourt Herbarium (UPH) were collected with the permission of the Curator and investigated in the Department of Plant Science and Biotechnology, University of Port Harcourt, Nigeria. This study was conducted from June, 2017 to July 2019. The information about the specimens studied is presented in Table 1.
Area of study: This research study was conducted in the Department of Plant Science and Biotechnology, University of Port Harcourt, Nigeria from June, 2017 to July, 2019.
Petiole anatomy: The petiole anatomy of the studied taxa was carried out by cutting a transverse section of the petioles of each of the taxa at the proximal, median and distal regions. The transverse sections were cut with the aid of a Reichert sliding microtome at a thickness of 8-15 μm. The sections were stained with safranin O and counter-stained in alcian blue, after which they were made to pass through different concentrations of ethanol for differentiation and dehydration. The sections were then mounted in a 25% dilute glycerin solution for examination under the microscope. Anatomical descriptions were made22,23. Photomicrographs of the different sections were made using a trinocular microscope (Amscope-T340B) fitted with a digital camera.
RESULTS
The results of current study’s findings on the anatomical characteristics of the cross-sections of the petiole at distal and basal portions of the taxa studied are presented in Table 2 and 3 and Fig. 1-5.
Vascular bundle arrangement and sizes in the petiole: Based on the number of vascular bundles (vb), size, arrangement, presence or absence of accessory vascular bundles (rb) and presence of interspersed vascular bundles (Int), they are categorized in the vascular system at the distal and basal portions of the Asteraceae species studied into 5 types namely:
| • | Type 1: Three large (major) vascular bundles in an open separate arc with two accessory vascular bundles in distal and base of the petiole in V. cinerea (Fig. 1n, 2n) and S. trilobata (Fig. 2j, 4j), E. alba (Fig. 1f, 3f) and E. aureus (Fig. 2e, 4f) and the petiolar base of A. africana, A. uliginosa, A. conyzoides, C. sumatrensis, E. pretamissa, S. nodiflora, T. procumbense, C. cinereum, A. hispidum, L. taraxacifolia, L.aurita, S. uliginosa, C. odorata, T. procumbense, S. nodiflora, S. filicaulis, A. houstonianum, L. taraxacifolia and S. costata (Fig. 1(a-c, e, g, j, l), Fig. 2(d, f, i, m, o), Fig. 3(d, j, l) and Fig. 4(c, g, i, l)), respectively |
| Table 1: | List of Voucher specimens studied |
| • | Type 2: Three large (major) vascular bundles in an open separate arc without accessory vascular bundles or the accessory vascular bundles occurring in the leaf-like wings in distal and base of the petiole in S. filicaulis (Fig. 2c, 4c) and at the petiolar base of C. crepidioides, A. conyzoides, C. sumatrensis, S. filicaulis, A. hispidum, C. crepidioides, L.aurita and S. uliginosa (Fig. 2k, Fig. 3c, e, Fig. 4c, k, m, o) |
| • | Type 3: Many vascular bundles including 3 large (major) ones in an open separate double arc with 1-3 accessory vascular bundles and interspersed vascular bundles in distal and base of the petiole in M. scandense (Fig. 1i, 3i) and petiolar base (basal portion) of C. odorata and T. diversifolia (Fig. 1d, 3k) |
| • | Type 4: The open separate arc having 5-7 vascular bundles of different sizes. Arc without accessory vascular bundles at petiole base of E. pretamissa (Fig. 3g) and at the distal and base of the petiole in V. amygdalina (Fig. 1m, 3g), arc with 1-2 accessory vascular bundles in E. ruderalis, V. biafrae and E. conyzoides (Fig. 1h, o, h), with 3-accessory vascular bundles in distal and basal portions of the petiole in W. trilobata (Fig. 2n, 4n) and petiolar base (basal portion) in V. biafrae (Fig. 3o) or with smaller interspersed vascular bundles petiolar base (basal portion) T. diversifolia, M. cordata, E. aureus, E. conyzoides and S. costata (Fig. 1k, 2b, f, g, l) and in distal portion in A. africana, E. ruderalis, V. conferta and C. cinereum (Fig. 3a, n, 4b, d) |
| • | Type 5: Vascular bundle forming a continuous open arc with accessory vascular bundles at both ends of the arc in distal and base of the petiole in V. conferta (Fig. 2a, 4a) |
The mean sizes of the vascular bundles at the distal portion of the petioles varied from 22.5 μm in Ethulia conyzoides to 97.5 μm in Erigeron aureus while at the basal end it varied from 22.5 μm in Sphagneticola trilobata to 80.8 μm in Melanthera scandense (Table 2, 3). Also, the thickness of the abaxial and adaxial parenchyma, adaxial and abaxial epidermises and the thickness of the petioles varied among the species studied and are diagnostic especially when combined with other anatomical features.
| Fig. 1(a-o): | Transverse section of basal portion of the petiole in Asteraceae studied, (a) A. africana, (b) A. uliginosa, (c) A. conyzoides, (d) C. odorata, (e) C. sumatrensis, (f) E. alba, (g) E. pretamissa, (h) E. ruderalis, (i) M. scandense (leaf-like wings), (j) S. nodiflora, (k) T. diversifolia, (l) T. procumbense, (m) V. amygdalina, (n) V. cinerea and (o) V. biafrae |
| rb: Rib trace, mb: Medullary vascular bundle, vb: Vascular bundle, tr: Trichome, sd: Secretory duct, Int: Interposed vascular bundles | |
| Fig. 2(a-o): | Transverse section of basal portion of the petiole in Asteraceae studied continued, (a) V. conferta, (b) M. cordata, (c) S. filicaulis, (d) C. cinereum, (e) A. hispidum, (f) E. aureus, (g) A. houstonianum, (h) E. conyzoides, (i) L. taraxacifolia (leaf-like wings), (j) S. trilobata (leaf-like wings), (k) C. crepidioides, (l) S. costata (m) L. aurita, (n) W. trilobata and (o) S. uliginosa |
| rb: Rib trace | |
| Fig. 3(a-o): | Transverse section of distal portion of the petiole in Asteraceae studied, (a) A. africana (leaf-like wings), (b) A. uliginosa, (c) A. conyzoides (leaf-like wings), (d) C. odorata, (e) C. sumatrensis (leaf-like wings), (f) E. alba (leaf-like wings), (g) E. pretamissa (leaf-like wings), (h) E. ruderalis (leaf-like wings), (i) M. scandense (Note: leaf-like wings), (j) S. nodiflora (leaf-like wings), (k) T. diversifolia (leaf-like wings), (l) T. procumbense (m) V. amygdalina, (n) V. cinerea and (o) V. biafrae (leaf-like wings) |
| rb: Rib trace, vb: Vascular bundle | |
| Fig. 4(a-o): | Transverse section of distal portion of the petiole in Asteraceae studied continued, (a) V. conferta, (b) M. cordata, (c) S. filicaulis (leaf-like wings), (d) E. aureus (leaf-like wings), (e) C. cinereum (leaf-like wings), (f) A. hispidum, (g) A. houstonianum (leaf-like wings), (h) E. conyzoides (leaf-like wings), (i) L. taraxacifolia (leaf-like wings) and (j) S. trilobata (leaf-like wings), (k) C. crepidioides, (l) S. costata (m) L. aurita, (n) W. trilobata and (o) S. uliginosa, |
| rb: Rib trace, vb: Vascular bundle, tr: Trichome | |
| Fig. 5(a-if): | Adaxial cuticle shapes of petiole in the taxa studied, (a) Undulating or wavy outline, (b-d) Deeply concave or slightly concave outline, (e) V-shaped outline, (f) Flat or relatively flat surface, (g) Slightly elevated with a relatively flat surface, (h) Convex surface and (i)-U-shaped surface |
Adaxial petiole outline: The surface of petiole of the species studied are densely hairy in A. africana, C. odorata, C. sumatrensis, E. ruderlis, M. scandense, T. diversifolia and V. conferta (Fig. 1a, d, e, h, i, k, Fig. 2a, Fig. 3a, h, i, k, 4a) and fairly/sparsely hairy or glabrous in the other species. The adaxial outline of the petiole of the different species investigated showed 8 different forms namely:
| • | Type 1 (Fig. 5a): Adaxial cuticle outline showed undulating or wavy outline in basal portion of A. africana, (Fig. 1), distal portions of W. trilobata, A. conyzoides, V. biafrae, M. cordata, A. houstonianum and L. taraxacifolia (Fig. 2n, Fig. 3c, o, Fig. 4b, g, i) and at both basal and distal portions of S. filicaulis and S. uliginosa (Fig. 2c, o, Fig. 4c, o) |
| • | Type 2 (Fig. 5b): Adaxial cuticle outline deeply concave in basal portions of A. uliginosa, S. nodiflora, C. crepidioides, S. costata (Fig. 1b, j, 2k, l) and distal portions of A. uliginosa and E. aureus (Fig. 3b, 4f) |
| • | Type 3 (Fig. 5c, d): Adaxial cuticle outline slightly concave in the basal and distal portions of E. alba (Fig. 1f, i), E. ruderalis (Fig. 1h, m) and M. scandense (Fig. 1i, 3i). This type of outline also occurred in A. conyzoides, C. odorata, C. sumatrensis, T. diversifolia, V. cinerea, A. houstonianum, E. pretamissa, S. nodiflora, T. procumbense, S. costata and W. trilobata (Fig. 1c-f, h, i, k, n, Fig. 2g, Fig. 3f-j, l, Fig. 4l, n) |
| • | Type 4 (Fig. 5e): Adaxial cuticle outline V-shaped in the basal portion of E. pretamissa, T. procumbense, A. hispidum and E. Aureus (Fig. 1g, l, Fig. 2e, f) and in basal and distal portions of S. trilobata (Fig. 2i, 4j) |
| • | Type 5 (Fig. 5f): Adaxial cuticle outline flat or relatively flat surface in basal and distal of C. cinereum (Fig. 2d, 4d) and basal petiolar portions of V. amygdalina, V. conferta, M. cordata, E. conyzoides, L. taraxacifolia and L. aurita (Fig. 1m, Fig. 2a, b, d, h, i, m) and in the distal petiolar portions of A. africana, C. sumatrensis, E. conyzoides and C. crepidioides (Fig. 3a, e, Fig. 4d, h, k) |
| • | Type 6 (Fig. 5g): Adaxial cuticle outline slightly elevated with a relatively flat surface in distal petiolar portions of T. diversifolia, V. cinerea and A. hispidum (Fig. 3k, n, 4e) |
| • | Type 7 (Fig. 5h): Adaxial cuticle outline convex in V. conferta (Fig. 4a) |
| • | Type 8 (Fig. 5i): Adaxial cuticle outline U-shaped in C. cinereum (Fig. 4n) |
Among the species studied, it was observed that some of them have leaf-like wings. The leaf-like wings were observed either at the distal or basal portions of the petiole or at both ends. This feature makes the species distinguishable and was found mainly at the distal portion of the petiole in most of the species studied, however, in some species like M. scandense, C. crepidioides, A. conyzoides and C. sumatrensis in Fig. 1i, 2k, 3c and e.
DISCUSSION
Studies on the vascular bundle arrangement and anatomical characteristics in Asteraceae are well documented2,3 however, comprehensive study on the petiolar vascular bundle arrangement is still lacking. Nevertheless, the number of vascular bundles, shape, arrangement and petiole outline are consistent in angiosperms and could be used in plant identification and classification24-26. Also, petiole anatomy including vascular bundle arrangement has been used in delimiting families such as Malvaceae25, Dipterocarpaceae24,26 and Annonaceae27. Furthermore, Noraini et al.28 used the petiole outline in the identification of some species of Coelotegia (Bombacaceae) and Olowokudejo29 in Cruciferae.
The vascular bundles in the species studied consist mainly of three large (major) ones and smaller (minor) ones. The vascular bundles in all the species studies formed an open separate arc except in V. conferta were the vascular bundle formed an open continuous/united arc (Fig. 2a, 4a). Remarkable among the family is the presence of 3 large (major) vascular bundles which is similar to those observed in Asteraceae2. Though the majority of the species have 3 main (lager) vascular bundles, few of them have more than 3 vascular bundles at either the distal or basal areas of the petiole. The 3 larger vascular bundles were observed more on the basal end of the petiole than the distal ends. M. cordata has 11 vascular bundles at both distal and basal ends but with 5 and 7 larger ones at the distal and basal ends, respectively. Also, V. brafrae and V. conferta have 5 and 1 larger vascular bundles each at the distal and basal ends, respectively. Furthermore, E. conyzoides and V. amygdalina have 4 and 6 larger vascular bundles at the distal ends, respectively (Table 2, 3). In other taxa, the vascular bundles are distinct numbering 3-7 without the smaller or minor ones. These observations were recorded in A. hispidum, A. conyzoides, C. crepidiodes and L. aurita which have 3 vascular bundles each. E. praetermissa has 5, S. filicaulis 3 and V. amygdalina (7) at the basal ends of their petioles. These differences made these species distinct from the remaining species studied. Among the taxa with three larger vascular bundles, there are variations in the number of smaller or minor ones. These variations in the number, size and arrangement of vascular bundles are genetically controlled30 and could be used in delimiting the taxa studied especially when combined with other characters such as the shape of the adaxial cuticle and vascular bundle type. At the genus level especially the taxa with more than one species like Ageratum, Spilanthes and Vernonia, we observed that the for among the species of Ageratum studied, the number of bundles and types at both distal and basal ends of the petiole are different but the outline of the adaxial cuticles are the same. Also, they have wing-like petioles. Similar observations were made for Spilanthes and Vernonia. Subsequently, at the family level, these features are specific to any genus, however, at the genus level, these features such as vascular bundle type and number are distinct and could serve as a diagnostic character among the taxa studied.
Notable among these is the presence of interspersed vascular bundles in A. africana, M. scandense, C. odorata and T. diversifolia. This feature was not observed among other members of this family studied and therefore made these species different from other members of this family studied. A similar observation has been made on members of Asteraceae2. Metcalfe and Chalk3 pointed out that petiolar anatomy in Asteraceae at the distal end has simple separate vascular bundles, varying in number and may be complex or simple. They found complex vascular bundles in some genera such as Arctium, Cirsium and Eupatorium (Chromolenea) and further noted that some genera have partly double arc of vascular bundles on the adaxial portion while some of the species are having separate vascular bundle arc accompanied by medullary strands, arc phloem around the inner edge of the xylem or petiole covered with thick cuticle or with leaf-like wings. Mainly the distal portions of the majority (73.33%) of the species studied have leaf-like wings while 76.67% of them have this structure at the basal portion. Some of the species studied like M. cordata, A. uliginosa, T. procumbens, C. odorata, V. amygdalina, V. conferta and C. cinereum have leaf-like wings at both basal and distal portions of the petiole while others have trichome at the edges of the petiole. The presence of these leaf-like wings has been reported major characteristics of Asteraceae3.
In this study, the petioles of the taxa studied have some close similarities however; some of the species could be differentiated from others based on this feature especially at the genus level. Generally, vascular bundle type 1 was found in two species viz. the distal part of A. africana petiole and the base of C. odorata petiole. Type 2 was found in a majority of the species such as distal and portions of E. alba, at the distal portion of A. conyzoides, E. pretamissa and V. cinerea and the base of C. conyza and S. nodiflora. The type 3 vascular bundle arrangement was only found in the distal portion and base of V. amygdalina. This feature however made this species distinct from all the other species studied. In the same way, vascular bundle type 4 was observed in the distal part of only two species (T. procumbense and V. cinerea ).
According to Noraini et al.28 petiole outline can be used for the identification of some species in genus Coelostegia (Bombacaceae). Another study on the petiole anatomy of selected species and genera in the family Cruciferae also has identified the differences of petiole outline on 46 taxa that can be used in taxonomic classifications29. Chia31 stated that the petiole outline in M. erythrocarpa and M. laurifolia are rounded in shape.
In this study, we observed eight (8) types of petiole outlines. Among the members of this family studied, we noted that the morphological and anatomical features of the petiole could be used to delimit them. Though some of the species studied had similar vascular bundles, the outline or cross-section and presence/absence of leaf-like wing are further used to distinguish them. Among the 24 genera and 30 species of Asteraceae studied, we observed five vascular bundle types and eight petiole outlines (cross-sections).
Among the vascular bundle types, Type-1 occurred in 19 species and 12 genera, type-2 in eight species and seven genera, type-3 in 3 species and three genera, type-4 in 12 species and 11 genera and type-5 in one species and one genus. The type-5 vascular bundle is restricted to V. conferta and is found at both distal and basal ends of the petiole in this species. Other ones occurred singly or in association with other vascular bundle type and are not restricted to a species. We noted that among the Spillanthes species studied, the vascular arrangement at the distal end of the petiole could be used to distinguish them. S. filicaulis had an only type-2 vascular bundle, S. costata had type-4 and type-1 vascular bundles and S. uliginosa had type-2 and type-1 vascular bundles. Also, in the genus Vernonia, they have different vascular bundles types but V. amygdalina and V. brafrae had a similar vascular type and could be further differentiated base on the cross-sectional outline of the petiole.
Also, the petiole outline type-1 occurred in 9 species, type-2 in 5 species, type-3 in 16 species, type-4 in 5 species, type-5 in 11 species, type-6 in 3 species, type-7 and 8 in 1 species each. Twenty three of the species had a leaf-like wing at either distal or basal end of the petiole while seven of the species do not have a leaf-like wing.
CONCLUSION
In this study, it is evident that the arrangement, number and sizes of vascular bundles in the petiole were fairly different and diagnostic especially the presence and number of accessory vascular bundles that varied among the species. Also, the presence and number of interspersed vascular bundles varied sparingly among the taxa especially at the genus and species level.
SIGNIFICANCE STATEMENT
This study discovers the importance of petiole anatomical information in the classification of Asteraceae that can be beneficial to diagnose the presence and number of accessory vascular bundles which shows variation. This study will help the researcher to uncover the critical areas of arrangement, number and sizes of vascular bundles in the petiole. Thus a new theory to find the number of interspersed vascular bundles may be arrived at.