Caladium bicolor (Aiton) Vent. belongs to the family Araceae and is characterized by various leaf colourations (Hutchinson and Dalziel, 1954). There are over 2000 cultivar names for Caladium, and over a hundred cultivars are  grown  today (Wilfret,  1993).  The  genus  Caladium comprises 12 species (Mayo et al., 1997), of which C. bicolor is the major source of cultivars. C. picturatum and C. marmoratum are now considered synonyms of C. bicolor (Madison, 1981). It is a monotypic genus that is   found  in  most  West  African  countries (Hutchinson  and Dalziel, 1954).

Source of materials

The materials for this study were collected from different parts of the country (Table 1), properly identified using Flora of West Tropical Africa (Hutchinson and Dalziel, 1954), and deposited at the University of Port Harcourt Herbarium. Live specimens are being maintained at the Ecological Center, University of Port Harcourt.

 

 

Petiole and midrib studies

Subsamples from the original materials (Table 1) were fixed in formalin, acetic acid and alcohol (FAA) for 12 h. Thereafter, the specimens were dehydrated in ethanol series of different concentration (30 and 50%) and stored in 70% ethanol until when needed. The leaf portion of the petiole, and the central portion of the midrib were sectioned following the method of Agbagwa et al. (2007). The sections were stained in Alcian blue and counter-stained with 1% Safranin red for two minutes, mounted on a slide, viewed and photographed with an Optika B-1000 FL LED microscope.

 

Epidermal studies

Foliar materials for epidermal studies were collected fresh from plants in the Ecological Centre. 0.5 to 1 cm square leaf cuttings were obtained from identical regions of each fresh leaf, generally from  mid-way  between  the leaf base and apex of lamina including the mid-rib. The adaxial and abaxial epidermal peels were obtained using sharp pointed forceps. Peels were stained with 1% safranin or alcian blue, rinsed with distilled water to remove excess stain and mounted in a drop of pure glycerol on clean glass slides. A cover glass was placed over the drop and sealed with nail vanish to prevent dehydration (Okoli and Ndukwu, 1992). The epidermal features that were observed include: organization of the epidermis, arrangement of the epidermal cells, nature of trichomes, shape of epidermal cells and nature of the anticlinal cell wall of the leaf epidermis, stomatal types, density and index. The stomatal index (SI) was determined based on Metcalfe and Chalk (1979), while the terminology for the stomatal type is taken after Malvey (2004).

Four variants of C. bicolor were identified. These specimens display a variety of colors including red, white, pink, yellow and green, and are tagged C. bicolor var. A, B, C and D (Figure 1). The coloration of the collections conformed to previous reports by Metcalfe and Chalk (1968). These authors reported that the genus Caladium in West Africa is monotypic with various colorations.

 

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Epidermal characteristics

No variation was observed on the shapes of the leaf epidermal cell among the different variants of Caladium studied. For instance, the shape of the upper (adaxial) and the lower (abaxial) epidermal surfaces in variant A, C and D are mainly pentagonal-hexagonal but rarely heptagonal, while the anticlinal cell wall are mainly straight and partly arced/curved. However, in variant B the shape of the adaxial and the abaxial epidermal surfaces are pentagonal-hexagonal (Figures 2 and 3, Table 2).

Variants A, B and D are amphistomatic (with stomata on both surface of the leaf) while variant C is hypostomatic (stomata on only one surface). This character makes this variant different from the other variants studied. The stomata types observed among the different Caladium variants are isotricytic, anisocytic, tetracytic, anomocytic and contiguous (Figures 2 and 3, Table 2). Among these stomata types, tetracytic and anomocytic stomata were dominant (Figures 2 and 3) and were observed on both leaf surfaces of variants A, B and D while in variant C they were recorded only on the lower surface (Table 2).  

 

 

 

 

Isotricytic and anisocytic occurred only on the lower leaf surface of variant D. The occurrence of the same stomata   type  among  these  variants   of   Caladium indicates very close phylogeny as shown in Sphenostylis stenocarpa (Hochst ex A. Rich) Harms by (Nyananyo and Osuji, 2007). The presence of isotricytic, anisocytic and contiguous stomata on variant D distinguishes it from the other variants. In the same vein, the presence of brachyparacytic stomata in the abaxial leaf epidermis of an accession of Xanthosoma ‘Ede Uhie’ and its  absence  in  other  cultivars  indicate divergent advancement in its evolution (Osuji and Nwala, 2015).

This suggests evolutionary divergence between Caladium variant D and the other variants. In a similar study among the Araceae, different stomata types namely anomocytic, actinocytic, paracytic, cyclocytic and transitionary types between paracytic and cyclocytic have been noted (Wang and Zhao, 2002). They observed that the epidermal cells are nearly isodiametric in outline with straight, arched and undulate anticlinal walls. Striate ornamentation occurs on periclinal walls of epidermal cells in some species. They noted that though the stomatal apparatus types in Araceae are of little taxonomical significance at intra-family level of Araceae, the combined characters of stomatal apparatuses, the shape of anticlinal wall and ornamentation of cuticules in guard cells may be useful for species identification (Wang and Zhao, 2002; Osuji and Nwala, 2015).

The stomata index (SI) varied from one variant to another. For instance, the stomatal index on the abaxial surface include- 25.93 to 37.5 (31.26±4.05), 22.22 to 37.50 (30.92±5.93), 6.25 to 36.36 (19.93±10.15) and 26.67 to 47.62 (36.64±7.68) for variants A, B, C and D respectively (Table 3). On the other hand, the stomatal index on the adaxial surface of the variants varied from 4.55 to 8.82 (6.31±2.24) in variant D to 6.25 to 8.11 (7.08±0.94) in variant A, and 4.35 to 11.76 (6.94±3.01) in B. The differences in stomata types, their distribution and indices on the leaf surfaces of these Caladium variants are diagnostic and could be used to distinguish among variants.

 

 

In this study, variants A and B have the same stomata types but the indices varied slightly from each other. Also, variant C has the same stomata types with A and B but it is hypostomatic while variant D has the same stomata types  with these variants in addition to isotricytic, anisocytic and contiguous stomata. This finding however conforms with Osuji and Nwala (2015) who noted the presence of stomata on both upper and lower epidermis of both Colocasia and Xanthosoma spp. They further noted that stomata were more on the lower epidermis than the upper epidermis. Also, they observed epidermal variations and differences in stomatal indexes within the cultivars of Xanthosoma and Colocasia and suggested that this could account for their ecological adaptation to variation in the degree of wetness of the environment. Also, stomata types and nature of the epidermal cells have been of diagnostic importance in other members of angiosperm namely Emilia (Ndukwu and Agbagwa, 2006), Abrus (Agbagwa and Okoli, 2006), Vernonia (Kemka-Evans et al., 2014) and Ixora (Essiett and Umoh, 2014).

 

Shape of midrib and petiole

The shapes of the adaxial surface of the midrib are relatively different from each other. In variants A (Figure 4a) and C (Figure 4h), the shapes are curved (convex); in variant B (Figure 4e), it is flat while in variant D it is relatively flat (Figure 4k). This character is fairly diagnostic among the different variants. The leaf lamina comprised one  layer  of  palisade   and spongy mesophylls  each (Figures 4b, f and i). The upper epidermal cells in the lamina of all the variants are isodiametric or oval. They elongate periclinally (Figures 4b, f and i). Also, the shapes of the petiole in all the variants studied are oval (Figures 5a, c, e and g). These similarities confirm that the variants have the same evolutionary origin and are of the same monotypic taxon (Metcalfe and Chalk, 1968); however intraspecific hybridizations may have produced the variants (Hayward, 1950; Birdsey, 1951; Wilfret, 1993; Deng and Harbaugh, 2006b).

 

 

 

 

 

 

 

 

 

 

 

 

 

Number of vascular bundle in the midrib and petiole

Variations in number and arrangement of vascular bundles in petiole and midrib have been used to differentiate species of the same genus or family (Ekeke and Mensah, 2015; Agbagwa and Ndukwu, 2004; Metcalfe and Chalk, 1979). For instance, in their studies, Ekeke and Mensah (2015) noted that the number of vascular traces in the midrib of members of Asteraceae varied from one species to another and is diagnostic. Also, Agbagwa and Ndukwu (2004) reported that the number of vascular bundles in the petioles of members of Cucurbita could be used to distinguish them (C. moschata, 10; C. maxima, 16 and C. pepo, 14).

In this study, the number of vascular bundles in the midribs  and  petioles  varied  from one variant to another (Table 3). The number of vascular bundles in the petiole of the different variants are variant A (19), variant B (27), variant C (22) and variant D (22).  In the midrib (Table 3), variant A has 19 vascular bundles, variant B (11), variant C (13) and variant D (23). This variation in the number of vascular bundles is diagnostic and could be used to distinguish the different Caladium variants. Though variants C and D had the same number of petiolar vascular bundle, the number of vascular bundle in their midrib differed and could be used to differentiate them. This finding therefore supports that the variation in number of vascular bundle in the petiole and midrib are diagnostic (Ekeke and Mensah, 2015; Agbagwa and Ndukwu, 2004; Metcalfe and Chalk, 1979) and however may suggest that intraspecific or interspecific hybridizations may have produced the variants.

 

 

Calcium oxalate types and sizes

Two main calcium oxalate crystal types were observed among the taxa studied. These are druses  and  raphides (Table 4, Figures 4 and 5). The druses are ubiquitous, raphides are predominantly found in the petiole while druses and raphides are found in the midrib. The occurrence of some of these crystals is specific or restricted to some tissues. For instance, among the taxa studied, the occurrence of the raphides is specific. They are found in the midrib and petiole but not in the lamina. This observation has been made in Dieffenbachia seguine (Araceae) (Gray, 2009). He noted that druses appear to be nearly ubiquitous throughout this plant occurring in most of plant parts except in the ovaries.

 

 

 

 

Furthermore, he observed that different portions of the same organ may have different crystal types such as the leaf margins which have a greater density of druses than the lamina, as well as overlapping raphide bundles not present in the lamina (Gray, 2009).

In the lamina, the calcium oxalates (druses) are embedded in the palisade and spongy mesophylls. They are identified by their dark and spiny nature. The accumulation of the calcium oxalate crystals in the leaf suggests that the taxa are poisonous. This explains why the leaves are not normally consumed by animals and humans except after being properly processed. These crystals could therefore be a source of defense to the plant against herbivores. This observation is in line with previous studies on Xanthosoma and Colocasia (Osuji, 2013; Uno et al., 2001).