Taxonomic significance of the vegetative anatomy of members of genera Colocasia ( L . ) Schott and Xanthosoma ( L . ) Schott in the family Araceae

Anatomical attributes are important for taxonomic studies of plants. This study investigated foliar and petiole anatomy of some members of the genera Colocasia and Xanthosoma. Similar and diagnostic characters critical for the taxonomy of the two genera were identified. The similar characters include, polygonal epidermal cell shape, straight adaxial anticlinal wall pattern, brachyparacytic stomata type, elliptic shaped stomata and unmodified raphide type. The presence of papillae on the adaxial surfaces of the members of genus Colocasia but not in the Xanthosoma taxa; lamellar collenchyma type in Xanthosoma mafaffa (Red), and unicellular non-glandular trichomes in Xanthosoma mafaffa (White) were recorded as diagnostic characters.

zones of the world (Croat, 1990).There are 3,750 species of aroids from 114 genera according to Petruzzello (2018), though Boyce and Croat (2018) reported 3,645 species from 144 genera.Members of these two genera, Colocasia and Xanthosoma, are important sources of food and medicine, especially in rural and poor communities where food security is posing a major challenge (Matemilola and Elegbede, 2017).
An interesting feature or character of aroids is their toxicity.Their cells possess calcium oxalate crystals in form of raphides and druses (Franceschi and Nakata, 2005;Arogundade and Adedeji, 2017).Therefore, Araceae members are enlisted as poisonous plants (Mulligan and Munro, 1990).Nevertheless people had devised methods for eliminating the poison to make these edible (Okiy, 1960;Ibe and Iwueke, 1984;Amanze, 2009).The corms and leaves of members of genera Colocasia and Xanthosoma have been part of delicacies around the world (Cable, 1984;Okeke, 1992, Amanze, 2009).
Morphological descriptions of these two genera have been studied (Purseglove, 1972;Burkill, 1985;Gill, 1988;Mayo et al., 1997;Ngoka, 1997;Bown, 2000), resolving some descriptive problems between the two genera.A ready distinction between Xanthosoma and Colocasia as ascertained by Burkill (1985), lies in the junction of the leaf lamina with the petiole.The leaf is attached to the petiole on the leaf margin in Xanthosoma, while in Colocasia, the attachment is towards the centre.That is, unlike the leaves of Colocasia, those of Xanthosoma are usually not peltate-the upper v-notch extends in to the point of attachment of the leaf petiole to the blade.
The genus Colocasia include six species of tuberous perennials from tropical Asia, grown there as a staple starchy food.Common names include Elephant-ear, Taro, Cocoyam, Dasheen, and Eddoe.Colocasia esculentum has between 28 and 42 chromosomes per cell that behave erratically during cell division.Thus, new permutations, resulting in new varieties are being formed without recourse to cross-pollination and seed production (Onwueme, 1978).Although cultivated plants rarely bloom, they are cultivated all the year round in subtropical and tropical areas.Taro is closely related to Xanthosoma and Caladium, which are commonly grown as ornamentals (Bown, 2000).
The genus Xanthosoma is a group of fleshy herbaceous stem-less plant with leaves arising from the crown of a central corm usually surrounded by a mass of cormels.A number of varieties exist based on yield, the colour of the flesh or skin, size of corm, storage quality, palatability and so on.The young leaves are eaten as a green vegetable (Kay, 1987).Mayo et al. (1997) reported chromosome number 2n = 22, 26, 39 and 52 for the genus.
This work is intended to shed more light on taxonomy of genera Colocasia and Xanthosoma which has been reported to be confusing (Green and Oguzor, 2009) using Arogundade and Adedeji 93 their leaf and petiole anatomical characters.

MATERIALS AND METHODS
Five taxa from the genera Colocasia and Xanthosoma were included in this work.The species and varieties are: C. esculentum var.esculentum (L.) Schott, C. esculentum var.antiquorum (L.) Schott, the red and white varieties of X. mafaffa Schott and X. saggitifolium Schott.The taxa were collected from different locations in the South Western part of Nigeria and were authenticated at Forestry Herbarium Ibadan (FHI), Oyo State and IFE Herbarium at Obafemi Awolowo University, Osun State, Nigeria.Voucher specimen was deposited at the IFE herbarium (Table 1).

Epidermal studies
The well expanded median portion of the leaf of each of the taxa was scrapped following the standard method described by Metcalfe (1960) and as adopted by Arogundade and Adedeji (2016).
Epidermal peels from both the adaxial and abaxial surfaces were obtained and afterwards stained with Safranin O.The peels were later mounted in dilute glycerine in readiness for microscopic examination.Features observed on the epidermal peels included the epidermal cell shape, the anticlinal wall patterns, stomata shape and size.The stomata area was calculated by multiplying the length and breadth of 50 stomata from at least five different plant accessions.Stomata indices of the adaxial and abaxial surfaces were calculated using the formula: S Stomata Index (I) = × 100 S + E Where S = Number of stomata and E = Number of ordinary epidermal cells plus the subsidiary cells in the same unit area.
Photomicrographs of the epidermis were taken for both the adaxial and the abaxial surfaces.

Petiole anatomy
A Reichert sliding microtome manufactured at Vienna, Austria NR. 367 019 was employed in cutting the transverse section of the petiole of the taxa at the proximal, median and distal regions.The sections were made at a thickness of 8 -15 µm.For staining, Safranin O and Alcian blue stains were used.After which differentiation and dehydration were carried out on the sections by passing them through varying percentages of ethanol (50%, 70%, 80%, 90% and absolute).The sections were later mounted in 25% glycerine and observed under the microscope Olympus XSZ-107BN binocular biological microscope manufactured by Zenith Laboratories, California.Photomicrographs of the different sections were taken.
All microscopic measurements were taken with the aid of an ocular micrometer inserted into the eyepiece of a microscope.They were later multiplied by the ocular constant with respect to the power under which they were taken in order to convert them to micrometer.

RESULTS
Tables 2 and 3 show the summary of the important    ) and stomata index ranges between 2.70 and 7.59% (mean = 4.75%).Papillae were present.

Abaxial epidermal characteristics
In C. esculentum var.antiquorum, epidermal cells were polygonal with straight anticlinal wall.They varied in size, shape and arrangement.Epidermal cell area ranges between 139.86 and 439.56 µm 2 (mean = 261.21µm 2 ).Brachyparacytic, occasionally anomocytic stomata types as well as two stomata sharing the same subsidiary cells, were observed.Stomata were elliptic in shape though occasionally circular (Plate 1C, D and E).Stomata size ranges between 149.85 -266.4 µm 2 (mean = 173.36µm 2 ) and stomata index ranges between 8.15 and 14.78% (mean = 10.51%).Papillae and druses are present.

Petiole anatomy
The summary of the proximal, median and distal regions of the petiole anatomy of the taxa studied is as shown on Table 5.

C. esculentum var. antiquorum
The outline of the adaxial surface of the proximal region was convex while that of the median and distal regions was concave.The outline is consistently round on the abaxial side of the petiole at the three regions.The epidermis was uniseriate and it was a single layer.One to four layers of parenchyma cells were encountered at the proximal and distal regions of the petiole, while one to three layers were identified at the median region.Lacunar collenchyma cells were encountered as discontinuous bundles on both the adaxial and abaxial sides of the petiole in all the three regions except in the distal region where it was not present on the adaxial side.Air spaces of varying diameter were present through the three regions of the petiole.Collateral vascular bundles were scattered throughout the ground tissue in the three regions with the xylem being consistently surrounded by one or two layers of xylem parenchyma.Unmodified and spindle-shaped raphides were present in the three regions of the petiole, starch grains were encountered in the median region while druses were found in both median and distal regions (Plates 2 to 4).

C. esculentum var. esculentum
The outline of the adaxial surface of the proximal region was convex, flat to slightly convex at the median region and concave at the distal region.The outline was consistently round on the abaxial side of the petiole at the three regions.The epidermis was uniseriate and it was a single layer.One to two layers of parenchyma cells were encountered at the three regions of the petiole.Lacunar collenchyma cells were encountered as discontinuous bundles on both the adaxial and abaxial sides of the petiole in all the three regions except in the distal region where it was not present on the adaxial side.Air spaces of varying diameter are present through the three regions of the petiole.Collateral vascular bundles are scattered throughout the ground tissue in the three regions with the xylem being consistently surrounded by a layer of xylem parenchyma.Spindle-shaped raphides were present in the three regions of the petiole, biforine raphides were observed only in the proximal region, unmodified raphide types and tannins were also observed in the distal region only while druses were present in both the median and distal regions (Plates 5 to 7).

Xanthosoma mafaffa (Red)
The outline of the adaxial surface of the proximal region was flat to slightly convex, convex at the median region and concave at the distal region.The outline was consistently round on the abaxial side of the petiole at the three regions.The epidermis was uniseriate and it was a single layer.Two to three layers of parenchyma cells were encountered at the proximal region of the petiole, while two to four layers were identified at the median and distal regions.Lamella collenchyma cells were encountered as discontinuous bundles on both the adaxial and abaxial sides of the petiole in all the three regions except in the distal region where it was absent on the adaxial side.Air spaces of varying diameter were present through the three regions of the petiole.Collateral vascular bundles were scattered throughout the ground tissue in the three regions with the xylem being consistently surrounded by one layer of xylem parenchyma.Unmodified raphides were encountered only in the median region of the petiole, starch grains were present in all the three regions, tannins were present only at the proximal region while druses were encountered at the median and distal regions (Plates 8 to 10)

X. mafaffa (White)
The outlines of the adaxial surface of the proximal and median regions were convex while that of the distal region was concave.The outline was consistently round on the abaxial side of the petiole at the three regions.The epidermis was uniseriate and it was a single layer.One to two layers of parenchyma cells were encountered at the proximal and median regions of the petiole, while one to three layers were identified at the distal region.Lacunar collenchyma cells were encountered as discontinuous bundles on both the adaxial and abaxial sides of the petiole in all the three regions except in the distal region where it was absent on the adaxial side.Air spaces of varying diameter are present through the three regions of

Xanthosoma saggitifolium
The outline of the adaxial surface of the three regions was convex while that of the abaxial side is consistently round.The epidermis was uniseriate and it was a single layer.One to six layers of parenchyma cells were encountered at the proximal and median regions of the petiole, while four to six layers were identified at the distal region.Lacunar collenchyma cells were encountered as discontinuous bundles on both the adaxial and abaxial sides of the petiole in the three regions.Air spaces of varying diameter were present through the three regions of the petiole.Collateral vascular bundles were scattered throughout the ground tissue in the three regions with the xylem being consistently surrounded by one layer of xylem parenchyma.Spindle-shaped raphides were encountered in all the three regions, while druses and starch grains were encountered in the median and distal regions (Plates 14 to 16).

DISCUSSION
Anatomical features have been found very useful in the classification of plant species and so the use of anatomical features in separating or delimiting species is germane.This is because most anatomical features are not affected or altered by the environmental factors.Anatomical research works that have been done on some species of Araceae, which include the works of Green and Oguzor (2009) on four selected species from genera Xanthosoma, Dieffenbachia and Colocasia in the South Eastern part of Nigeria; Ina and Eka (2013) on leaf surface comparison of some species in the genera Alocasia, Colocasia and Remuatia in Indonesia; Osuji and Nwala (2015) on some cultivars of Xanthosoma and Colocasia also in the South Eastern part of Nigeria, were limited to the epidermal studies of the selected species or cultivars only.This study provided some more detailed information on the epidermal surfaces of more taxa as well as the details of the transverse sections of the petioles in the three petiole regions, proximal, median and distal regions.
Similar and diagnostic characters which were useful tools in the taxonomy of the two genera were identified in this study.The epidermal cell shape on the adaxial and abaxial surfaces of the two varieties of Colocasia studied; C. esculentum var.antiquorum and C. esculentum var.esculentum as well as that of the three taxa in the genus Xanthosoma; X. mafaffa (Red), X. mafaffa (White) and X. saggitifolium is polygonal.This was a common character to the two genera.Anticlinal wall pattern on the adaxial surface was straight in all the taxa of Colocasia and Xanthosoma studied.On the abaxial surface however, though the wall pattern was straight in all the taxa, it was straight to wavy in X. saggitifolium.This separated X. saggitifolium from the two varieties of X. mafaffa.Adedeji et al. (2007) also employed anticlinal wall pattern in the separation of some species in the family Solanaceae.Generally, there were more stomata on the abaxial surfaces than on the adaxial surfaces of all the taxa studied.The presence of brachyparacytic stomata complex type in all the taxa agrees with the findings of Osuji and Nwala (2015) who reported the presence of paracytic and brachyparacytic stomata types in the cultivars of Xanthosoma and Colocasia that they studied.However, additional stomata complex types were encountered on one or both surfaces of some of the taxa studied.On the adaxial surface, anisocytic stomata were found in C. esculentum var.esculentum which separated it from the other variety, C. esculentum var.antiquorum.
On the abaxial surface, anomocytic stomata were the additional stomata encountered in all the taxa but not in the red variety of X. mafaffa.According to Hetherington and Woodward (2003), stomata types are genetically determined and so cannot be influenced by the environment.
Stomata size is also quite diagnostic (Thair and Rajput, 2009).The largest stomata sizes were encountered in X. saggitifolium while the smallest sizes were encountered in C. esculentum var.antiquorum.Elliptic shaped stomata were common to all the taxa studied; notwithstanding, some of the taxa have additional circular shaped stomata especially on their abaxial surfaces.Stomata index was generally higher on the abaxial surface than on the adaxial surface in all the taxa studied.Druses of calcium oxalate crystals were found on the adaxial and abaxial epidermal surfaces of C. esculentum var.antiquorum only.This distinguished it from C. esculentum var.esculentum and the members of the genus X. in this study.Adedeji and Illoh (2004) also reported a separation among some species of Hibiscus based on the presence of druses.The presence of globular or spherical papillae in some of the taxa studied is of diagnostic value.Papillae were encountered on the adaxial surfaces of the two varieties of Colocasia studied, that is, C. esculentum var.antiquorum and C. esculentum var.esculentum but not on those of the three taxa of Xanthosoma.This can be employed in the delimitation of the members of the genus Colocasia from the members of the genus Xanthosoma.On the abaxial surface, however, they were encountered in all the species and varieties of the two genera.They were of variable shapes and sizes.The subsidiary cells in most of these taxa have no papillae.Osuji and Nwala Plate 11.Transverse section of petiole and cell inclusions in the proximal region of X. maffafa White variety.A. Abaxial outline (×40).B. Adaxial outline (×40).C & D. Petiole transects (×100 &×400 respectively).E. Unmodifiedraphide (×400).F. Unmodified raphide and druses (×400, Arrowed).G. Druses (Arrowed) and starch grains (×400).H. Vascular bundle (×400).
(2015) also reported the presence of papillae on the abaxial surfaces of the X. mafaffa accessions but did not report it on the two surfaces of Colocasia.Osuji (2006) separated two species in the genus Musa based on the presence or absence of papillae.
Ridges or folds of the cuticle form ornamentations on them; these ornamentations largely consist of striae, hence, striated cuticle (Metcalfe and Chalk, 1979).
According to Solereder (1908), these striations are very useful for specific diagnosis and are not always developed in the same way on the two surfaces of the leaf, they are also taxonomically stable.Adedeji and Illoh (2004) used cuticular striations to separate some species of Hibiscus.In this study, striated cuticle was found on the adaxial surface of the red variety of X. mafaffa as well as on the abaxial surfaces of all the taxa of the genus Xanthosoma studied, that is, the red and white varieties of X. mafaffa and X. saggitifolium.However, the striation pattern on the abaxial surface of the red variety of X. mafaffa revealed a unique feature as it formed a continuous network with the papillae in such a way that it kept the stomata and epidermal cells out of focus.This is a delimiting factor among the members of the genus Xanthosoma.Also, no cuticular striations were observed in the two varieties of C. esculentum.
The results of the anatomy of the petiole have provided a wide range of characters that were equally of diagnostic value in delimiting the Araceae species studied.Thakuri and Patil (2011) have separated some species of the family Euphorbiaceae using petiole anatomy.Concave adaxial petiole outline in the median region of C. esculentum var.antiquorum differentiated it from C. esculentum var.esculentum and the three members of genus Xanthosoma in this study with convex or flat to slightly convex outline.In the distal region, however, convex adaxial petiole outline separated X. saggitifolium from all the other taxa where concave adaxial petiole outline was observed.The cells of the epidermis in the petiole of all the taxa studied were of one layer and uniseriate.Parenchyma cells of varying number of layers from one to six were present in the proximal, median and distal regions of the petiole of the taxa studied.
All the taxa studied have the collenchyma cells as discontinuous bundles but the types and location of Plate 14. Transverse section of petiole and cell inclusions in the proximal region of X. saggitifolium. A. Abaxial outline (×40); B. Adaxial outline with Tannins (Circled) (×40); C and D. Petiole transects (×100 &×400 respectively).E, F and G. Spindle-shaped raphides (×400); H. Vascular bundle (×400).collenchyma cells can be employed in separating them.Lacunar collenchyma cells were encountered in the two varieties of Colocasia; C. esculentum var.antiquorum and C. esculentum var.esculentum, as well as X. mafaffa (White) and X. saggitifolium whereas the collenchyma cells were the lamellar type in Xanthosoma mafaffa (Red) which separated it from the white variety and the other taxa.The collenchyma cells were observed on both the adaxial and abaxial surfaces of the proximal and median regions of all the taxa in this study.The variation observed was in the distal region where it occurred on both surfaces of X. saggitifolium and only the adaxial surfaces of the other four taxa.Collateral vascular bundles scattered in the ground tissues were found in the three regions of all the taxa studied and all the xylem cells were surrounded by xylem parenchyma.Vascular bundles are known to be scattered in the ground tissues of Monocots, which is one of their major characteristics (Fahn, 1974).
Raphides, druses, tannins and starch grains were observed in petiole of all the taxa.The presence of raphides and druses which are calcium oxalate crystals have been well established among the members of the family Araceae (Middendorf, 1982;Mayo et al., 1997;Arogundade and Adedeji, 2016).From the work of Keating (2004), eight types of raphides have been established.They are unmodified, styloids, wide cells (a form of the unmodified raphide crystal), elongated cells, tubular cells, articulated tubes, spindle-shaped, and biforine raphides.The unmodified raphide type was observed in all the taxa studied.The spindle-shaped type was observed only in the two varieties of Colocasia and X. saggitifolium while the biforines were observed only in C. esculentum var.esculentum.This can also be employed in delimiting the taxa in this study.Tannins were observed in C. esculentum var.esculentum and X. mafaffa (Red).They were not encountered in the other taxa.Where present, they were always found close to the vascular bundles.Aroids, plants in the Araceae family are generally known to be glabrous.Trichomes are highly unusual in their leaves and stems (Solereder and Meyer, 1928;Mayo et al., 1997;Bown, 2000).Interestingly, in this study, some trichome-like structures were found on the adaxial side in the distal region of the petiole of X. mafaffa (White).They were more or less unicellular, nonglandular trichomes.Solereder and Meyer (1928) also reported the presence of unicellular hairs in X. pubescens.This obviously separated X. mafaffa (White) from its Red variety and the other taxa in this study.

Conclusion
In conclusion, the members of genera Colocasia and Xanthosoma can be separated based on their anatomical Arogundade and Adedeji 105 features although many of the observed characters are common to them affirming their familial classification.Some other characters cut across the two genera.The unifying features observed include polygonal epidermal cell shape, straight adaxial anticlinal wall pattern, brachyparacytic stomata, elliptic shaped stomata and unmodified raphide type.Diagnostic features that can be employed in separating the two genera are the presence of papillae on the adaxial surfaces of the two varieties of Colocasia which were not observed on the adaxial surfaces of the Xanthosoma taxa.Also cuticular striations observed only in the members of genus Xanthosoma but not in the members of genus Colocasia.Worthy of note is the presence of unicellular non-glandular trichomes observed only in X. mafaffa (White).

Plate 1 .
Leaf epidermal surfaces of taxa of Colocasia and Xanthosoma.A and B. Adaxial epidermis of lamina of C. esculentum var.antiquorum; C, D and E. Abaxial epidermis of lamina of C. esculentum var.antiquorum; F. Adaxial epidermis of lamina of C. esculentum var.esculentum; G and H. Abaxial epidermis of lamina of C. esculentum var.esculentum; I and J. Adaxial epidermis of lamina of X. mafaffa (Red Variety).K and L.; Abaxial epidermis of lamina of X. mafaffa (Red Variety); M. Adaxial epidermis of lamina of X. mafaffa (White Variety); N. Abaxial epidermis of lamina of X. mafaffa (White Variety); O. Adaxial epidermis of lamina of X. saggitifolium; P. Abaxial epidermis of lamina of X. saggitifolium.
Transverse section of petiole and cell inclusions in the proximal region of C. esculentum var.esculentum. A. Abaxial outline (×40); B. Adaxial outline (×40).C and D. Petiole transects (×100 and ×400 respectively).E. Spindle-shaped (×400).F. Biforine raphides (×400).G.Vascular bundle (×400) the petiole.Collateral vascular bundles were scattered throughout the ground tissue in the three regions with the xylem being consistently surrounded by one layer of xylem parenchyma.Unmodified raphides were encountered in the proximal and median regions, druses and starch grains were present in the three regions of the petiole.Trichome-like (eglandular, uniseriate trichomes) structures were identified in the distal region of the petiole (Plates 11 to 13).

Table 1 .
Voucher specimen numbers of the taxa at IFE herbarium.

Table 2 .
Summary of important qualitative foliar epidermal characteristics of the adaxial surfaces of the taxa studied.

Table 3 .
Summary of important qualitative foliar epidermal features of the abaxial surfaces of the taxa studied.

Table 4 .
Summary of the quantitative foliar anatomical parameters.

Table 5 .
Summary of the proximal, median and distal regions of the petiole anatomy of the taxa studied.