Leaf epidermal and petiole anatomical features as taxonomic characters in some Vernonia species in Nigeria

Foliar and petiole micro-morphological characteristics of some members of the genus Vernonia namely; Vernonia amygdalina Del. (bitter variety), Vernonia amygdalina Del. (non-bitter variety), Vernonia cinerea (L) Less., Vernonia galamensis (Cass.) Less., and Vernonia adoensis Sch. Bip . in Jos, Nigeria was investigated to provide additional micro-morphological characters to support existing taxonomic information regarding the species. Epidermal peels of fresh leaves were made and transverse sections of petio les were cut using a rotary microtome. Staining was done using Safranin and slides observed using a light microscope. Quantitative characters were measured and subjected to Duncan Multiple Range Test. Stomatal distribution was amphistomatic except for V. galamensis and V. cinerea with hypostomatic distribution; stomata type was mostly anomocytic except V. galamensis with paracytic stomata. Stomata index varied among taxa with V. adoensis and V. amygdalina (bitter variety) having the highest (10.84 %) and lowest (0.67%) respectively. Anticlinal cell wall patterns were straight to slightly undulate while wavy anticlinal cell wall pattern was diagnostic to V. cinerea . Trichome types observed were glandular and multicellular uniseriate with highest and lowest trichome indices recorded in V. adoensis (2.80%) and V. galamensis (0.02%) respectively. Petiole outline was convex to concave, epidermis was uniseriate, trichome multicellular uniseriate and vascular bundles bicollateral. Variations observed in the stomatal complex, epidermal cell complex, petiole outline, petiole vasculature type, trichome and stomata indices could be employed for species identification and delimitation.


INTRODUCTION
The use of anatomical data has been long recognized in plant taxonomy since variations within taxa is usually ref lected in anatomical f eatures. Data f rom gross morphology, wood anatomy, and f oliar epidermal morphology have proved valuable in plant identif ication and classif ication (Kadiri et al., 2006). One of the noteworthy taxonomic practices is f oliar epidermis, and characters such as trichomes, stomatal f eatures and anticlinal cell wall patterns have been used in taxonomic studies of a numb er of f amilies (AbdulRahaman et al., 2014). The signif icance of petiole anatomical characters in understanding the relationship within plant taxa has also been studied and reported to be usef ul in separating plants of dif f erent species, genera and f amilies (Metcalf e andChalk, 1979, Kamel andLoutf y, 2001). Its usef ulness as a supportive tool in plant taxonomy has recently increased and progress has been made in using this data for plant classif ication (Ogundare andSaheed, 2012, Bercu andPopoviciu, 2014).
The genus Vernonia contains more than 500 species distributed in North and South America, tropical Af rica especially in Nigeria, Zimbabwe and South Af rica and it is domesticated in parts of West Af rica (Farombi, 2003, Kemka-Evans et al., 2017. Some Vernonia species are consumed as vegetables while others are used in indigenous medicine. For instance, the roots and leaves of Vernonia amygdalina are used in treatment of malaria, inf ertility, diabetes, gastrointestinal problems and sexually transmitted diseases (Ene andAtawodi, 2012, Adedapo et al., 2014). The roots and leaves of Vernonia cinerea are also used f or treating nerve disorder, kidney disease, and as a potent analgesic (Syed et al., 2011, Thiagarajan, 2014. A decoction of Vernonia cinerea is used in treatment of urinary tract inf ection, cough, stomach ache and diarrhea (Farombi and Owoeye, 2011). In Nigeria, the leaves of V. amygdalina induces f ertility in women (Adedapo et al., 2014), and in Ghana, it is consumed for breast milk enhancement in nursing mothers (Kankara et al., 2015) Researchers have attempted the taxonomy of the genus Vernonia and some of these studies f ocused on the f oliar morphology (Oladele, 1990, Adedeji and Jewoola, 2008, Nwakanma et al., 2018. However, a comparative study on the petiole anatomy of species of this genus have not received much attention and data is still insuf f icient. Theref ore, this study describes the epidermal morphology and petiole anatomical structures of f our species of Vernonia and the non-bitter variety, with the aim of providing additional micro-morphological characters to support existing taxonomic inf ormation regarding the species.

Sample collection.
Freshly collected specimens of Vernonia were collected in Jos, Plateau state specif ically in Federal college of Forestry and Shere hills (Latitudes 0 93 N and 9 0 95 N and longitudes 8 0 89 E and 8 0 93 E

Sample Preparation and Analysis
The epidermal peels of both the adaxial and abaxial surf aces of the leaves of each plant were obtained. Specif ically, the leaf materials were soaked in concentrated trioxonitrate (v) acid (HNO3) in a glass petri dish, covered and allowed to stand f or 3 hours. The epidermises were separated with f orceps, rinsed in distilled water, stained in Saf ranin solution and mounted in 5% diluted glycerol (Metcalf e and Chalk, 1978). The parameters of the stomata observed were stomatal distribution, stomatal index, stomatal length and width. Epidermal cell length and width were measured. The characters were on the basis of 25 sample size and the measurement for each character was replicated 10 times. The epidermal characteristics were described using Metcalf e and Chalk (1978). Parameters of the trichome determined include trichome type, trichome index, trichome length and width. Stomata and trichome indices were calculated using the f ormulae of Stace (1965). Where, S=Stomata number per unit area E=Epidermal cell number per unit area T=Trichome number per unit area For the petiole anatomy of the species, transverse sections f rom the median regions for each of the species were obtained using a rotary microtome. The sections were stained in 10 % aqueous Saf ranin solution and mounted in 5% diluted glycerol Specimen slides were observed using light microscopy and photomicrographs were captured under magnif ication of X400 and X100 using Olympus microscope with attached Celestron digital camera. The identif ication and description of tissue and cell f ollowed Fahn (1997). The quantitative data was subjected to Analysis of Variance and Duncan Multiple Range Test (Duncan, 1955, Bailey, 1976.

Petiole anatomy
Petiole outline was convex on abaxial surf ace and concave on adaxial surf ace. Epidermis was uniseriate having multicellular uniseriat e trichomes. Collenchyma oval to angular, 5-8 layered and parenchyma oval, 7-15 layered ( Table 2). Vascular bundle bicollateral f orming an arc with about 9 bundles. Crystals and druses were randomly distributed on the cortex. Petiolar vasculature type was closed ( Fig. 2C-D).

Foliar epidermis
Epidermal cells were pentagonal to hexagonal and anticlinal wall was straight on the abaxial surf ace (Fig. 3B); mean length and width of cells up to 3.64 μm and 2.62 μm respectively. Stomata paracytic, stomata index 2.96 %, mean stomata length and width up to 2.46 μm and 1.01 μm respectively. Glandular and multicellular uniseriate trichomes present, trichome index 2.28 %, mean trichome length and width 11.86 μm and 4.06 μm respectively. However, on the adaxial surf ace (Fig. 3A), epidermal cells were pentagonal to hexagonal with undulating anticlinal walls; mean length and width of cells up 3.32 μm and 2.03 μm respectively. Multicellular uniseriate trichome present, trichome index 0.02 %, mean trichome length and width were 10.74 μm and 3.07 μm respectively (Table 1). Stomata are absent

Petiole anatomy
Petiole outline was convex on the abaxial surf ace and concave on adaxial surf ace. Epidermis was uniseriate having multicellular uniseriat e trichomes. Collenchyma oval to angular, 3-4 layered and parenchyma oval to angular, 5-7 layered (Table 2). Vascular bundle bicollateral, C shape with about 6 bundles. Crystals and druses were randomly distributed on the cortex. Petiolar vasculature type was closed ( Fig. 4C-D).

Petiole anatomy
Petiole outline was convex on the adaxial surf ace and concave on abaxial surf ace. Epidermis was uniseriate having multicellular uniseriat e trichomes. Collenchyma oval to angular, 4-5 layered and parenchyma oval, 9-10 layered ( Table 2). Vascular bundle bicollateral, C shape with about 5 bundles. Crystals and druses were randomly distributed on the cortex. Petiolar vasculature type was closed ( Fig. 5C-D).

Foliar epidermis
Epidermal cells were irregular and anticlinal wall was wavy on the abaxial surf ace (Fig. 6B); mean length and width of cells up to 3.46 μm and 3.03 μm respectively. Stomata anomocytic, stomata index 9.58 %, mean stomata length and width up to 1.54 μm and 0.66 μm respectively. Glandular trichome present, trichome index 1.23 %, mean trichome length and width 6.86 μm and 3.18 μm respectively. However, on the adaxial surf ace (Fig. 6A), epidermal cells were irregular with wavy anticlinal walls; mean length and width of cells up 3.82 μm and 4.22 μm respectively. Stomata was absent on the adaxial surf ace. Multicellular uniseriate trichome present, trichome index 0.97 %, mean trichome length and width were 24.64 μm and 4.22 μm respectively (Table 1).

DISCUSSION
Anatomical characters have been employed in the classif ication of plant taxa and its usef ulness in the delimitation and separation of species is reported relevant. Foliar epidermal anatomy is one of the most noteworthy taxonomic characters f rom the systematic point of view and several delimitations of taxa have been done based on leaf epidermal characters (Aworinde et al., 2014, Ekeke and Agbagwa, 2015, Bello et al., 2017, Raza et al., 2020. Mbagwu and Edeoga (2006) opined that characters such as stomata type, distribution and size, epidermal cell shape, trichome type and distribution are tools usef ul in the identif ication and classif ication of many angiosperms. These anatomical characters are reported not to be altered or af f ected by the environmental f actors.
Diagnostic anatomical characters usef ul in identif ication and delimitation of these species were observed in this study. Generally, the appearance of stomata on the leaf surf aces varied among the studied species. The presence of hypostomatic f eature in V. cinerea and V. galamensis contradicts earlier reports by Nwakanma et al. (2018) and Asuzu (2020) who reported amphistomatic f eature in the two species. Further, the presence of paracyctic stomata type in V. galamensis also contradicts the observations of Asuzu (2020) that reported only anomocytic stomata in the species studied. However, this f eature distinguishes V. galamensis f rom others with anomocyctic stomata type. Hetherington and Woodward (2003) opined that stomata types cannot be inf luenced by the environment as they are genetically determined and are theref ore good taxonomic tool. Further, the number of stomata was more on the abaxial surf ace than the adaxial surf ace. Consequently, the stomata index was higher on the abaxial surf aces of the taxa studied. There was an overlap in stomata size (length and width) of the taxa which limits its usef ulness as a diagnostic f eature. This agrees with Davies and Heywood (1963) who reported that stomata size is too variable to be considered a diagnostic character.
The presence of irregular cells and wavy anticlinal wall pattern on both surf aces of V. cinerea distinguished it f rom others with polygonal shaped cells and slightly undulate to straight anticlinal wall pattern.   A range of petiole anatomical characters was observed and some of these characters are diagnostic and important f or the delimitation of these Vernonia species. The presence of uniseriate epidermis, multicellular uniseriat e trichomes, oval to angular collenchyma cells and bicollateral vascular bundles in the petioles of all the taxa studied could serve as diagnostic f eatures f or the genus and this corroborates the report of Asuzu (2020) who observed similar characters in the Vernonia species studied. There are however dif f erences in petiole outline, petiolar vasculature type, number of vascular bundles, number of layers of parenchyma and collenchyma cells which could be employed for species identif ication. The number of vascular bundles ranges f rom 5 to 9 with V. adoensis having the highest number of vascular bundles. The petiolar vasculature type was open in V. cinerea and V. galamensis and closed in the other three taxa. The dif f erences in these f eatures have been reported by Akinnubi et al. (2013) to be usef ul in delimitation and classif ication of some species in the f amily Asteraceae.

CONCLUSION
The importance of f oliar and petiole anatomical characters in some Vernonia species in Nigeria was shown in this investigation. Taxonomic similarities and dissimilarities among the species and diagnostic characters important f or species identif ication and delimitation were shown. These diagnostic characters include stomata size, stomata index, stomata types, trichome index, petiole outline, petiole vasculature type and epidermal cell shapes, which varied among the species of this genus. Although, this study agrees with some of the existing taxonomic data on the f oliar epidermis and petiole anatomy of this genus, it also shows characters not previously reported, thereby serving as additional data to already existing taxonomic inf ormation on these species.

Conflict of interest
Authors have no conf lict of interest to declare.