The six species of the genus Citrus namely: C. sinensis, C. limon, C. aurantifolia, C. paradizi, C. reticulate and C. maxima belong to dicotyledonous family Rutaceae. The family has over 150 genera and 1500 species. They are mainly distributed in both tropical and temperate regions; especially in Australia and South Africa. In West Africa, it is represented by 9 genera and about 30 species. Several important genera such as Citrus, Aegle, Murraya, Ruta, Africana, Limnocitrus, Citropsis, Fortunella, Glycosmis, Micromelum, and Fagara are major representatives of the family. The family comprises of mainly trees and shrubs, rarely herbs. Some shrubs are climbers and xerophytic. The stems are erect, sometimes climbing, thorny branched, woody or rarely herbaceous, cylindrical, solid, green when young while grey when old. Leaves are single or pinnate or palmate. Compound exstipulate. They are usually alternate and rarely opposite. The leaves are dotted with glands which contain volatile oil. This volatile oil gives the leaves its typical smell. Leaves are reduced to spine in early stages of genera such as Citrus and Feronia sp. The petiole is glabrous to pubescent. Inflorescence may be cymose or racemose. Flowers are solitarity, axillary, bisexual, actinomorphic or occasionally zygomorphic, white or yellow hypogynous. Sometimes, the flowers are unisexual e.g. Evodia, Zanthoxylum, etc.

Calyx four to five sepals, polysepalous, green in colour. The corolla is polypetalous. Petals are coloured white, yellow or red. Androecium contains eight to ten stamens. Filaments are free with bilobed anther. Gynoecium is syncarpous, contains three, four or five carpels. Placentation is axile. The style is short and decidous. Fruits vary in different genera. They may be berry, drupe, capsule, samara or follicle.

Some genus such as Citrus is used for medicinal purposes. They contain Vitamin A,B,C and P as well as flavonoids, sugar, organic acids and mineral salts. Orange diminishes blood viscosity and produces a protective effect on blood vessel due to vitamin P, among other substances [1]. In some cultures, genus such as Citrus is used for traditional purposes. During China New Year Festivals, Mandarn Orange and Tangerines are considered traditional symbols of abundance and good fortune. They are often decorated and presented as gifts to friends and relations, even business associates [2]. Citrus also serve as ornamental trees. Orange flowers have delicious scent and are used in decorations and banquets [3]. The leaves of Ruta graveolens, Fegara budrunga, Murraya koenigii and Toddalia asiatica are used as condiments and for flavouring curries. The tender shoots of F. oxyphylla are used as vegetables. Some are used as fodder (Aegle marmelos and Feronia limonia). Citrus serves as source of raw material to juice industrials. Naringin (a flavour) and neohesperidin dihydroahalcone from grape fruit and pummelo (Shaddock) have application as artificial sweetners. Timbers are yellowish, hand and close-grained, used in cabinet work, turnery and marquetry. The trees are used for afforestation of dry areas since the Citrus species have a water conserving system [4].

Plant families known for their wide diversity and complexity constitute many problems to biosystematicists using traditional methods based on gross morphology to characterize the species under the large family. In most recent times, leaf epidermal features as anatomical characters have received very considerable attention by Taxonomists [5-7].

The application of epidermal characters such as shape of epidermal cells, wall pattern, type and arrangement of stomata, stomatal index, stomatal aperture, size and types of trichomes, row of cell per trichome are some of the important systematic characters used in modern biosystematic studies. The importance of epidermal characters in plant taxonomy can be appreciated from review of works carried out on some plant species by various researchers [8]. Edeoga and Ikem [9] used epidermal morphology of some species of Boerhavia to establish interspecific relationships among the different species they investigated. Stace (1965b) was able to review the tribal, generic and specific characters of the family Combretaceae based on their leaf epidermal morphological characters. Mbagwu [10] studied leaf epidermal features of eight Vigna species and separated V. gracellis and V. racemosa from other species based on trichome morphology. Edeoge and Okoli [11] in Dioscoreaceae, Edeoga and Ogbebor [12] in Commelinaceae; Nyananyo and Olowokudejo in Portulaceae; Inyama et al [13] in Compositae; Gill and Nyawuama [14] in Labiatae were all workers who have used leaf epidermal features to solve are problem or the other in these families. Inyama et al. [15] used the epidermal morphology of three species of Chinpophyllum to establish intraspecific relationships among the taxa investigated. According to researchers, epidermal and cuticular traits of plants can serve as useful tools exploitable in the systematic of the present day Angiosperms [5,10,16].

Due to the complex taxonomic and systematic nature of the genus Citrus, there is need for revalidational studies on the interspecies relationship among the genus. Till now, only morphological features have been the bases of classifying the genus. There is little or no known work on epidermal, thus the need to use this evidence to revalidate six selected species of the genus Citrus. This study shall therefore reassess the taxonomic identities of these taxa using epidermal feature for proper biosystematic data collection. Also, the study shall try to establish inter specie relationship among the taxa investigated.

Specimen collections

The studies were made on living plant specimens. The specimen of the six species of the genus Citrus namely: C. sinensis, C. limon, C. aurantifolia, C. paradisi, C. reticulata and C. maxima were collected from Ministry of Agriculture and Natural Resources Nekede, Agricultural Development Programme (ADP) farms, Plant Garden at Aladinma Housing Estate, Homestead Garden at Amakohia Layout, Songhai Farms, Nekede, Imo ADP Egbeada and Homestead, Obazu Mbieri.

The sample specimen were identified and authenticated by authorities at Imo State University Herbarium were deposited at Imo State University Herbarium with Herbarium No. IMSUH 001-006. The study was conducted at the Plant Science and Biotechnology laboratory in Imo State University, Owerri, Nigeria.

Epidermal features

Fresh leaves from each of the six taxa namely, C. sinensis, C. limon, C. aurantifolia, C. reticulata, C. paradisi and C. maxima were washed in tap water. The leaves were painted with clear finger nail polish on both surfaces and allowed to dry. After drying, pieces of short clear cellophane tape were firmly pressed over the dried nail polish on the surfaces. The tapes were carefully peeled from the leaves and affixed on clean slides, stained with 1% ethanol safranin for one minute and temporarily mounted in aqueous glycerol solution and observed under. Ten slides were prepared per species and the type of stomata and shape of epidermal cells was determined according to Van Cotthem [17]. Also five out of ten slides were selected per species for the counting of the number of stomata, epidermal cells and other epidermal features. Frequency/Stomatal index per unit area were made from the five slides selected per species. The unit area used was the microscopic field of view at x 10.

Then four different counts were made from different portions of each slide. The thickness of the cell wall, type of trichome and the subsidiary cells were also determined. Data for the counts were organized and presented in Tables 1-2. Photographs illustrating the epidermal features were taken from the slides using Leitz Wetzler Ortholux microscope fitted with Viviter-v-335 camera (Figures 1-3). The stomatal index (S.I.) per specie was calculated as shown below:

Table 1: Epidermal studies of six Citrus species studied (upper epidermis).

Table 2: Epidermal studies of six Citrus species studied (lower epidermis).

Where S=number of stomata

E=number of epidermal cells

The anticlinal walls of the epidermis are straight and thick-walled at both upper and lower epidermis in all the species of Citrus studied. Epidermal cells are flat and variedly shaped in each species studied in the upper epidermis. It is rectangular to cuboidal to pentagonal to trapezial in C. sinensis; rectangular to triangular to hexagonal to trapezial in C. limon; rectangular to hexagonal to pyramidal to trapezial in C. aurantifolia; rectangular to pyramidal to trapezial in C. reticulata; hexagonal to cuboidal to trapezial in C. paradisi and rectangular to cuboidal to hexagonal to trapezial in C. maxima while in the lower epidermal, the epidermal shape shows variation, being rectangular to hexagonal to pyramidal in C. sinensis; rectangular to cuboidal to hexagonal in C. limon; rectangular to cuboidal to trapexial but rectangular to cuboidal to pyramidal to trapezial in C. reticulata, C. paradisi and C. maxima, distinguishing themselves from the rest of the epidermis. In the upper epidermis, the number of epidermis shows consistent variation, ranging from 320-335 in C. sinensis; 302-309 in C. limon; 349-362 in C. aurantifolia; 490-505 in C. reticulata; 405-419 in C. paradisi and 452-468 in C. maxima while in the lower epidermis the consistency persisted ranging from 258-267 in C. sinensis, 235- 246 in C. limon; 280-295 in C. aurantifolia; 389-399 in C. reticulata; 367-378 in C. paradisi and 342-360 in C. maxima. The epidermal cell length ranges from 15-22.5 μm in C. sinensis; 12.5-27.5 μm in C. limon; 13.75-22.5 μm in C. aurantifolia; 12.5-20 μm in C. reticulata; 15-32 μm in C. paradisi and 12.5-17.5 μm in C. maxima in the upper epidermis indicating that there is an affinity between C. sinensis and C. paradisi; and among C. limon, C. aurantifolia, C. reticulata and C. maxima while in the lower epidermis the epidermal cell length ranges from 12.5-22.5 μm in C. sinensis; 12.5-25 μm in C. limon; 12.5-20 μm in C. aurantifolia; 12.5-22.5 μm in C. reticulata 12.5-22.5 μm in C. reticulata and 10-17.5 μm in C. paradisi, inferring closeness among C. sinensis, C. aurantifolia and C. reticulata and between C. limon and C. paradisi; C. maxima distinguishing itself from others. The epidermal cell width in the upper epidermis is 7.5-13.75 μm in C. sinensis; 7.5- 17.5 μm in C. limon; 7.5-12.5 μm in C. aurantifolia; 6.25-12.5 μm in C. reticulata; 7.5-13.75 μm in C. paradisi and 3.75-12.25 μm in C. maxima. Affinity is indicated among C. sinensis, C. limon, C. aurantifolia and C. paradisi, showing distinctiveness in C. reticulata and C. maxima while in the lower epidermis, the epidermal cell width ranges from 5-10 μm in C. sinensis; 2.5-10 μm in C. limon; 5-7.5 μm in C. aurantifolia; 6.25- 12.5 μm in C. reticulata; 5-7.5 μm in C. paradisi and 6.25-10 μm in C. maxima showing that C. sinensis, C. aurantifolia and C. paradisi are related on one hand, while C. reticulata and C. maxima are related on the other hand, whereas C. limon distinguished itself from others. All the six species are hypostomatic with anomocytic type of stomata. The epidermal carpet architecture shows no appendages (trichome) or subsidiary cells in all the species. Sheath cells, modified epidermal cells are found covering the vein areas, prominent in C. sinensis, C. limon, C. paradisi and C. maxima in the upper epidermis whereas in the lower epidermis, they are prominent in C. limon, C. aurantifolia and C. reticulata. The number of stomata per unit area ranges from 64-70 in C. sinensis; 60-70 in C. limon; 55-66 in C. aurantifolia; 14-26 in C. reticulata; 40-50 in C. paradisi and 60-7 in C. maxima. C. sinensis, C. limon and C. maxima share the same affinity with highest number of stomata per unit area, followed by C. aurantifolia and C. paradisi but C. reticulata having the lowest number of stomata per unit area. This feature followed almost the same trend with the stomatal index, the highest being in C. limon with 21.13 and least in C. reticulata with 4.7 while it is 20.46 in C. sinensis; 17.59 in C. aurantifolia; 10.53 in C. paradisi and 15.70 in C. maxima. The length and width of stomata showed consistent variations being 5-9.5 μm and 2.5-7.5 μm in C. sinensis; 5-7 μm and 2 -5 μm in C. limon; 6.25-8.75 μm and 4.5-5 μm in C. aurantifolia; 7.5-12.5 μm and 5-7.5 μm in C. paradisi; 5.25-10 μm and 3.75-6 μm in C. maxima, showing relationship between C. sinensis and C. limon; C. aurantifolia and C. maxima; and between C. reticulata and C. paradisi. Contiguous stomata showed their presence in C. sinensis, C. limon, C. aurantifolia and C. maxima while absent in C. reticulata and C. paradisi. The mega stomata are present in C. sinensis and C. limon but absent in other species, showing affinity between C. sinensis and C. limon. (Tables 1 and 2; Plates 1-2).

Stomata in all the taxa studied are anomocytic and hypostomatic. On the lower epidermis, the anomocytic stomata observed in these taxa agreed with the findings of Mbagwu, et. al., [18] who reported same types of stomata in C. limon, C. Sinensis; C. auarntifolia and C. maxima which are four of the six species considered. On the contrary, the result disagreed with Obiremi et al (2001) who reported paracytic stomata in C. limon and C. auarntifolia; paracytic to anomocytic in C. simensis and C. reticulate. The presence of anomocytic stomata have been reported by some authors in different angiosperms. Shah and Gopal [19] in Discoreasceae; Edeoga and Ikem [9], and Mbagwu and Edeoga [20] in Vigna species. Presence of contignous stomata distinguished four of the taxa C. sinensis, C. limon, C. aurantifolia, and C. maxima from C. reticulate and C. paradise. This is the first time of reporting contignous stomata in the genus Citrus. The absence of sheath cells observed in C. sinensis and C. paradise is also a differentiating feature to separate C. rericulata from C. paradise. In the species were they occurred; it was observed that the sheath cells are found mostly above or covering the vein areas. This could be a mechanism to withstand excessive turgor pressure along the vessels during fluid condition as survival strategy. While the presence of mega stomata reported in C. senensis and C. limon can be used to distinguish C. sinensis from C. paradise. These mega stomata are usually surrounded by larger epidermal cells. Based on the shape of epidermal cells, rectangular, hexagonal, pyramidal shaped epidermal cells observed in C. sinensis definitely distinguished it from the rest of the taxa studied. While C. reticulate, C. paradise and C. maxima showed strong affinity based on the shape of epidermal cell walls. Rajiagopal, et al. [21] in Ternera; Gill and Ngawuame [14] in Bicarpellatea plants, and Patel and Shah [22] in Chilli were some researchers who used shape of epidermal cell as taxonomic tool in differentiating different taxa they studied.

Other features observed such as straight anticlinal and thick celled walls, support the interspecie relationships among the species studied. The taxa studied could be distinguished based on stomatal index as Edeoga et al. [9] on Boerharia. The stomata index showed that C. sinensis and C. limon are closely related; C. aurantifolia and C. maxima are closer while C. reticulate and C. paradisi can be differentiated from each other and the other taxa studied. C. reticulate having the lowest stomata index is easily differentiated from all the taxa. It was observed that C. aurantifolia has high stomata index than C. reticulata. This is contrary to Obirim et al. [4] who reported high stomatal index in C. reticulate and lowest stomatal index in C. aurantifolia.

On the upper epidermis, the shape of epidermal cells varies in each taxa and presence of rectangular and trapezium shaped cells in all but one specie indicating the close affinity between the taxa studied. The presence of Pentagonal shaped cells separated C. sinensis from the rest of the taxa while the absence of rectangular shaped epidermal cell distinguished C. paradisi from the other taxa studied. The number of epidermal cells also varies per species and can be used to delimilate the taxa [23-26].

The sheath cells were observed to occur in four of the taxa but absent in two of the taxa: C. aurantifolia and C. reticulata. Also, the presence of sheath cells on both the upper and lower epidermis of C. limon delinate it from the other taxa studied. Therefore, similarities observed on the epidermis of the six Citrus taxa studied strongly supported the relationship while the difference established their placement into different species [27,28].

The use of epidermal features of the taxa studied is of great taxanomic importance in supporting both their phylogenetic relationship of the taxa and their treatment as different species, but for a more detailed taxonomic study there is need to extend this type of taxonomic study to other evidences such as cytology, histochemistry, morphology, etc. before taking final taxonomic decision of the six investigated taxa.