Foliar anatomy of some species of Asteraceae in South Western Nigeria

The foliar anatomy of 12 species of Asteraceae around Ile Ife in South Western, Nigeria was described. The distinguishing characteristics of taxonomic value include; venation patterns, areole shapes, number of veinlet endings, trichome types, arrangement of vascular bundles. Venation types are actinodromous, craspedodromous or camptodromous, the presence of rectangular areoles are predominant in the species studied and this show family characteristics. However, the presence of crystal druses in the areoles of C. odorata is of diagnostic importance. The type of vascular bundle in the leaf midribs is classificatory as it divided the taxa studied into two groups; vascular bundles are amphicribal in Bidens pilosa, Chromolaena odorata, Launaea taraxacifolia, Crassocephalum crepidiodes, Tridax procumbens and Vernonia cinerea and bicollateral in Ageratum conyzoides, Aspilia africana, Emilia praetermissa, Synedrella nodiflora, Tithonia diversifolia and Vernonia amygdalina.


INTRODUCTION
Asteraceae (Compositae) is a very large cosmopolitan family whose members are highly advanced.It belongs to the sub-class Asteridae in the order Asterales.Asteraceae is the second largest family in the division Magnoliophyta with 1,100 genera and over 20,000 recognized species (Ming, 1999).The majority of Asteraceae species are herbaceous although an important component of the family consists of shrubs or even trees, many plants in the family Asteraceae are economically important as weeds, ornamentals, medicinals and green vegetables (Olorode, 1984).
The angiosperms are endowed with macromorphological characters of significant taxonomic values which can be easily observed with the naked eye or simple hand lens.Morphological attributes of vegetative organs have often constituted the mainstay of taxonomic studies in plants (Polhill, 1968;Pilbeam and Bell, 1979;Adedeji, 2005) and are very important in classification.The use of anatomical methods in taxonomic investigations cannot be over emphasized.Although no character is absolutely immutable, but some are more fixed than the others and it is on those that are less plastic that the systematic anatomist rely.Taxonomic decision based on epidermal studies are quite reliable because they are not really affected by environmental conditions (Barthlott, 1981), thus comparative plant epidermal studies have been found to be reliable in taxonomy and systematics (Stace, 1969;Ogunkunle and Oladele, 2000;Metcalfe andChalk, 1950, 1979;Naik and Nigrude, 1981;Palmer and Tucker, 1981;Adedeji, 2004;Adedeji and Illoh, 2004) all the authors have all stressed the taxonomic importance of anatomical features which along with other characters form taxonomic, identification and classification of plants.
The present study reported the use of foliar anatomy in establishing the taxonomic relationships between twelve species of Asteraceae.
These portions were decolorized by boiling in 90% ethyl alcohol to remove chlorophyll, and treated in 2% sodium hydroxide solution over night.They were rinsed in water thrice and transferred into 5% domestic bleach (JIK).They remained in the bleach until they became completely white, then rinsed in water thrice and preserved in 50% ethanol as described by Olatunji (1983).These were stained in 1% aqueous solution of Safranin O and mounted on a clean slide in 25% glycerol and the edges of the cover slips sealed with nail vanish.

Sectioning
Transverse sections of the leaf were cut at 20 µ thickness using Reichert Sledge Microtome and best sections preserved in 50% ethanol.The sections were stained in 1% aqueous solution of Safranin O for 5 min washed in 3 changes of water to remove excess stain and counterstained in 1% solution of Alcian blue for 5 min then washed in three changes of water and dehydrated by passing through series of ethyl alcohol: 50, 70, 80, 90 and 100% with two changes in 100% alcohol (dehydration process), and excess stain (differentiation process).The dehydrated and differentiated sections were cleared in xylene to remove last trace of water, to clear the sections (making it more transparent) and to remove last traces of ethanol and since xylene is the solvent of the mountant (DPX) used, it prevent cloudiness of the slide.The sections were mounted in DPX mountant according to Akinloye et al. (2012).Photomicrographs of the leaf sections of the twelve species were taken with Amscope digital camera attached to a light microscope.All microscopic measurements were made with the aid of an ocular and stage micrometer.The drawings were prepared in the scientific illustration unit of the National History museum, Obafemi Awolowo University by the museum's scientific illustrator with about twenty eight years experience in scientific illustrations.The slides were observed directly under LEICA DM500 binocular light microscope using different magnifications, the drawings were prepared without the aid of Camera Lucida but with painstaking replication to scale of the arrangement of the cells and structures in pencil line drawings before being transferred unto tracing films using Rotring pen size 0.1 with black Rotring ink.The drawings were later scanned into the computer for insertion into the appropriate sections in the text.

Herbarium survey
All plant species used in this work were collected authenticated in the Ife Herbarium and voucher specimens deposited in the same herbarium (Table 1).

Ageratum conyzoides Linn.
Venation is actinodromous, perfect, marginal, basal.Areoles arrangement is random.The shape is triangular, quadrangular and pentagonal; 38.4 -86.3±1 µm long and 24.9 -92.3±1 µm wide.The veinlet endings are singly divided and number ranges between 0 -2.Lamina (Plate 1A and B): Epidermis uniseriate, cells rectangular.The cuticle on both surfaces are not prominent and where they are seen, they are non-striated.Palisade mesophyll one layer thick, consisting of cylindrical shaped parenchyma cells that are closely packed.Mean length of palisade mesophyll is up to 28.9±1.80 µm.Spongy mesophyll consist of parenchyma cells that are largely irregular in shape, irregularly arranged with intercellular spaces.Simple uniseriate multicellular trichome present.Midrib (Plate 1C and D): Epidermis uniseriate, the epidermal cells are irregular, occasionally polygonal.Vascular bundle, 1, bicollateral.Simple uniseriate multicellular trichome present.

DISCUSSION
A survey of literature of leaf anatomy shows that data obtained from it can be used amply for the clarification of taxonomic and phylogenetic relationships.The commonly used characters like palisade and spongy features, types of vascular bundles, trichome types were largely employed in this study.In all the species studied, the major or primary veins are pinnate.Venation types were actinodromous, craspedodromous or camptodromous, the presence of rectangular areoles were predominant in the species studied and this show family characteristics.However, the presence of crystal druses in the areoles of C. odorata is of diagnostic importance.The veinlet terminating end per areole also varies from one species to another, ranges from 0-2 in A. conyzoides, A. africana, C. crepidioides, E. praetermiissa L. taraxacifolia, S. nodiflora and V. amygdalina.For these taxa, number of veinlet endings is classificatory though there is an overlap.The veinlet endings divided singly or bifurcated.The general significant difference observed in the quantitative characters may be used to separate the species.C. odorata has a higher areolar area than the other species while V. cinerea has a higher number of veinlet endings than others (Plate 13A, B, C and D).
Leaf anatomy according to Carlquist (1961) provides a variety of features that could be used for taxonomic purposes.Many researchers have utilized leaf anatomical features for taxonomic consideration in many species of plants.These include the works of Naik and Nigrude (1981) on Chlorophytum, Ogundipe and Olatunji (1991) on Cochlospermum, Illoh (1995) on Celosia, Adedeji (2004) on Emilia and Adedeji and Illoh (2004) on Hibiscus.Though there is uniformity in the anatomy of the leaf in the species studied, characters like the shape of palisade and spongy mesophyll cells, non-prominence and non-striation of the cuticles and types of vascular bundles could be used to classify members of the family.The type of vascular bundle in the leaf midribs is classificatory as it divided the taxa studied into two groups; vascular bundles are amphicribal in B. pilosa, C. odorata, L. taraxacifolia, C. crepidiodes, T. procumbens and V. cinerea and bicollateral in A. conyzoides, A. africana, E. praetermissa, S. nodiflora, T. diversifolia and V. amygdalina.Similarly, the shape of vascular bundle is classificatory in those with dissected xylem, that is, A. africana and C. crepidiodes and those without dissected xylem, this delimit the two from other species studied.Among all the taxa studied, S. nodiflora is quite distinct in the surface view of the epidermis by having deep groove Venation patterns in the species studied.A, Ageratum conyzoides; B, Aspilia africana; C, Bidens pilosa; D, Chromolaena odorata; E, Crassocephalum crepidiodes; F, Emilia praetermissa; G, Launaea taraxacifolia; H, Synedrella nodiflora; I, Tithonia diversifolia; J, Tridax procumbens; K, Vernonia amygdalina; L, Vernonia cinerea; AR, Areole; DR, crystal druses; VE, veinlet endings.(A-D): Leaf anatomy in Ageratum conyzoides; (A and B): Transverse section leaf (lamina); (C and D): Transverse section leaf (midrib).BTR-Bicellular trichome, EP-Epidermis, PM-Palisade mesophyll, SM-Spongy mesophyll, ST-Stomata, SUBTR-Simple uniseriate bicellular trichome, SUMTR-Simple uniseriate multicellular non -glandular trichome, VB-Vascular bundle.(E-H): Leaf anatomy in Aspilia africana (E and F): Transverse section leaf (lamina); (G and H): Transverse section leaf (midrib).BTR-Bicellular trichome, EP-Epidermis, PM-Palisade mesophyll, SM-Spongy mesophyll, ST-Stomata, SUBTR-Simple uniseriate bicellular trichome, SUMTR-Simple uniseriate multicellular non -glandular trichome, VB-Vascular bundle.

Table 1 .
IFE Herbarium (Osun state, Nigeria) voucher specimen number for the twelve Asteraceae species studied.
Crystal druses are present and prominent.Lamina (Plate 4A and B): Epidermis uniseriate, cells rectangular.The cuticles on both surfaces are not prominent, where they seen, they are non-striated.Palisade mesophyll one layer thick consisting of tightly packed cylindrical elongated parenchyma cells.Mean length of palisade mesophyll up to 27.5±1.80µm.Spongy mesophyll consist of parenchyma cells largely irregular in shape, irregularly arranged with intercellular spaces.Simple uniseriate bicellular and multicellular trichomes are present.Midrib (Plate 4C and D): Epidermis uniseriate, epidermal cells are irregular variable in size and arrangement.Vascular bundle, 1, amphicribal, shield shaped.Trichome: Simple uniseriate multicellular trichome are present.
(Plate 9A and B): Epidermis uniseriate, cells are irregular varying in sizes and arrangement.Cuticles are not prominent, non-striated.Palisade mesophyll one layer thick, tightly packed