MOLECULAR SYSTEMATICS AND ETHNOBOTANICAL STUDIES ON THE GENUS Crotalaria L. IN NORTHERN NIGERIAN SAHELIAN SAVANNAH: A CASE STUDY OF KATSINA STATE SAMAILA YARADUA SAMAILA (B.Sc. BIOLOGY) MSC/15/BIO/0016 A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOLOGY, UMARU MUSA YARADUA UNIVERSITY, KATSINA, IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN BIOLOGY JULY, 2017 DECLARATION PAGE I hereby declare that this work is the product of my own research efforts; undertaken under the supervision of Dr. Abubakar Bello and has not been presented and will not be presented elsewhere for the award of a degree or certificate. All sources have been duly acknowledged. ______________________________ SAMAILA YARADUA SAMAILA MSC/15/BIO/0016 CERTIFICATION PAGE This is to certify that the research work for this dissertation and the subsequent preparation of this dissertation by “Samaila Yaradua Samaila MSC/15/BIO/0016”were carried out under my supervision. Head of Department Dr. Abdulhamid Ahmed Sign and Date: _____________________ Major Supervisor Dr. Abubakar Bello Sign and Date: _____________________ Co Supervisor Dr. Akilu Sada Sign and Date: _______________________ APPROVAL PAGE This research has been examined and approved for the award of the degree of MASTERS OF SCIENCE in Biology (Plant Biosystematics and Taxonomy). External Examiner …………………………………… Internal Examiner …………………………………… Major Supervisor …………………………………. Co Supervisor …………………………………. Head of Department ……………………………………. Representative of the Postgraduate School …………………………………… ACKNOWLEDGEMENTS In the name of Allah, the most beneficent, the most merciful. All praise is to Allah, the lord of mankind, who gave me the strength and ability to conduct this research. His blessing and salutations be upon his Prophet Muhammad (S.A.W), his progeny, his companions and his followers up to the Day of Judgment. I profoundly extend my sincere appreciation and gratitude to my supervisor Dr Abubakar Bello for his efforts and support towards the completion of this work despite his tight schedules when he was away for his post doctoral fellowship in South Africa, no words are enough for expression of my profound gratitude to him may Allah grant him and his family Aljannutul fiddaus ameen. I also thank Dr Akilu Sada for his role as Co Supervisor and Dr Sulaiman Sani Kankara for providing guidance and advice. I proudly extend my sincere appreciation and gratitude to the post graduate coordinator Dr. Nasir Hassan Wagini, head of department Dr. Abdulhamid Ahmed and the entire staff of biology department of Umaru Musa Yaradua University, Katsina for their elder and fatherly advice, support and concerned on me towards my studies. My sincere gratitude goes to my parents Late Samaila Labaran Yaradua and Hajiya Hindatu Gagare, May there soul rest in perfect peace ameen, for their moral and spiritual support they gave to me and to my Grand Mother Hajiya Rabi Isah Kaita and Hajiya Talle for their concerned and prayer to my success, my sister and brothers Isah Samaila Yaradua, Hadiza Samaila Yaradua and Sadiq Samaila Yaradua for their love and care. May Allah grant them Jannatul Firdaus, ameen. I will also like to express my gratitude to all my classmates, relatives for their contribution towards my studies. I sincerely thank Umaru Musa Yaradua University, Katsina for funding my studies and department of Biology, Umaru Musa Yaradua University, Katsina. DEDICATION This research is dedicated to my late parents, Samaila Labaran Yaradua and Hajiya Hindatu Gagare. May their soul rest in perfect peace ameen. TABLE OF CONTENT Content Page Title page ………………………………………………………………….. i Declaration page …………………………………………………………... ii Certification page …………………………………………………………. iii Approval page …………………………………………………………….. iv Acknowledgements ……………………………………………………….. v Dedication ………………………………………………………………… vii Table of contents …………………………………………………………. viii List of tables ………………………………………………………………. xii List of figures ……………………………………………………………… xiii List of plate ……………………………………………………………….. xiv Abstract ……………………………………………………………………. xv CHAPTER ONE: INTRODUCTION The Genus Crotalaria L …………………………………………………… 1 History of classification ………………………………………………...... 2 Morphometrics …………………………………………………………….. 4 ITS Nuclear Ribosomal DNA…………………………………………….. 5 Statement of the problem ………………………………………………….. 6 Justification ……………………………………………………………...... 6 1.7 Aim and objectives …………………………………..……………………. 7 CHAPTER TWO: LITERATURE REVIEW 2.1 Taxonomic history of the family Fabaceae ……………………………….. 8 2.2 Taxonomic history of the tribe Crotalarieae……………………………...... 9 2.3 Taxonomic history of the genus Crotalaria……………………………….. 12 2.4 Infrageneric Classification in the genus Crotalaria……………………….. 15 2.5 Ethnobotany ……………………………………………………………….. 21 2.6 Economic importance of Crotalaria ………………………………………. 26 CHAPTER THREE: METHODOLOGY 3.1 Study Area ………………………………………………………….…...... 28 3.2 Taxon sampling …………………………………………………………… 29 3.3 Morphometric analysis ……………………………………………………. 29 3.3.1 Data analysis ………………………………………………………………. 32 3.4 Molecular study …………………………………………………………… 33 3.4.1 DNA extraction ………………………………………………………….... 33 3.4.2 Polymerase Chain Reaction (PCR) ……………………………………….. 34 3.4.3 Agarose Gel Electrophoresis ……………………………………………… 35 3.4.4 Gel Extraction ……………………………………………………..………. 36 3.5 Phylogenetic analysis …………………………………………………........ 37 3.6 Ethnobotanical Data ………………………………………………………. 37 3.6.1 Data analysis ……………………………………………………………..... 38 3.6.1.1 Relative Frequency of Citation (RFC) …………………………………….. 38 CHAPTER FOUR: RESULTS AND DISCUSSION 4.1.1 Morphometric result ………………………………………………………. 39 4.1.1.1 Clustering ………………………………………………………………….. 39 4.1.1.2 Ordination …………………………………………………………………. 40 4.1.2 Species Identification ……………………………………………………… 43 4.1.3 DNA Extraction, Gel Electrophoresis……………………………………… 44 4.1.4 Phylogenetic Analysis …………………………………………………….. 44 4.1.5 Taxonomic Revision and Description …………………………………….. 47 4.1.6 Ethnobotany ………………………………………………………………. 55 4.2 Discussion …………………………………………………………………. 58 CHAPTER FIVE: SUMMARY, CONCLUSION AND RECOMMENDATIONS 5.1 Summary ………………………………………………………………...... 63 5.2 Conclusion ………………………………………………………………… 65 5.3 Recommendations ………………………………………………………… 65 References ………………………………………………………………… 66 Appendix ………………………………………………………………..... 78 LIST OF TABLES Table Page Table 2.1: Sectional and subsectional classification for Crotalaria by Polhill (1968, 1982), Bisby and Polhill (1973), and Ansari (2008) ……………………………………19 Table 2.2: The infrageneric classification system from the crotalaria since 1973 (Le Roux et al., 2013) …………………………………………………………………………......20 Table 3.1: List of species within the genus Crotalaria taken for the study ……………30 Table 3.2: List of qualitative and quantitative characters and character states used in morphometric analysis ………………………………………………………………....31 Table 3.3: Primers used in PCR reactions ……………………………………………...35 Table 4.1: Similarity matrix based on Jaccard’s coefficient ……………………………40 Table 4.2: Loadings of the first and second components in the PCA …………………42 Table 4.3: Collections of Crotalaria …………………………………………………...43 Table 4.4: List of medicinally utilised species …………………………………………56  LIST OF FIGURES Figure Page Figure 1.1: rDNA ITS structure …………………………………………………………... 5 Figure 2. 1. A cladogram showing the relationships of the tribe Crotalarieae ……………. 11 Figure 3.1: Map of Katsina showing the study area ……………………………………… 28 Figure 4.1. UPGMA phenogram resulting from cluster analysis …………………………. 39 Figure 4.2: Plot of the first two principal components (PCs) in principal component analyses (PCA) ……………………………………………………………………………................ 41 Figure 4.3: PCA loading of the characters ………………………………………………… 42 Figure 4.4 Phylogenetic relationships of Crotalaria (43 taxa) based on Bayesian Inference analysis of ITS region ……………………………………………………………………… 46 LIST OF PLATE Plate page Plate 4.1: Result of gel electrophoresis of the 7 species ………………………………….. 45 Plate 4.2: vegetative and flower morphology of C. macrocalyx ………………………….. 48 Plate 4.3: Vegetative, flower and fruit morphology of C.spp …………………………… 49 Plate 4.4: Vegetative, fruit and flower morphology of C. senegalensis …………………. 50 Plate 4.5: Vegetative, fruit and flower morphology of C. spp ……………………………. 51 Plate 4.6: Vegetative, fruit and flower morphology of C. spp ……………………………. 52 Plate 4.7: Vegetative, flower and fruit morphology of C. pallida var. obovata ………….. 53 Plate 4.8: Vegetative, flower and fruit morphology of C. pallida ……………………....... 54 Plate 4.9: Vegetative morphology of C. retusa …………………………………………... 55 ABSTRACT Molecular systematic and ethnobotanical studies of the genus Crotalaria in the Northern Nigerian sahelian savannah: a case study of Katsina state was conducted to determine their phylogenetic relationships and their positions in the current infrageneric system of classification. I also revised the taxonomy of the species in the study area and document their ethnobotanical uses. A field survey was conducted, where 7 different species and one variety were collected and analyzed using morphometric analysis. The result showed that all the collected species are distinct at Euclidian distance of 0.2 in the cluster analysis. Four out of the seven species were identified as Crotalaria pallida, C. retusa, C. senegalensis and C. macrocalyx. The variety was identified as Crotalaria pallida var. obovata. The cophenetic correlation value (r) which was found to be 0.964 showed that all the species are closely similar. The ordination analysis based on the result of the PCA separated the specimens into 7 groups corresponding to the result of cluster analysis. The first two component of the PCA account for 81.5%. The results also revealed that leaf morphology contributed a lot in delimination of the species with length of leaflet having 0.7 and leaflet width having 0.40 on the PCA loading respectively. In determining phylogenetic relationships, DNA was extracted from each of the 7 species, and the nuclear ribosomal DNA gene (ITS) region of genomic DNA was PCR amplified using ITS4 and ITS5 primers. Sequencing reaction was done with the PCR product after purification. The data were analysed using bayesian inference analysis in MrBayes. The results showed that four (C. macrocalyx, C. retusa, C. sp, and C. pallida) of the seven species sampled were distributed across the various sections of Crotalaria (Hediriocarpae clade, Calycinae clade, and Stipulosae clade respectively), while three, Crotalaria sp., Crotalaria sp. and Crotalaria sp. were distributed across (Incanae clade, Longirostres clade respectively). Crotalaria macrocalyx and Crotalaria sp. are more closely related (PP, 0.99), Crotalaria restusa and Crotalaria sp. are also closely related. All the seven species are closely related and they belong to the genus Crotalaria (PP, 1.00). The results of the ethnobotanical data obtained using semi structured questionnaire showed that the species have good medicinal value in the treatment of various common ailments such as skin infection, fever and ulcer. Crotalaria retusa is reported to have high medicinal uses with RFC value of 0.41.  CHAPTER ONE INTRODUCTION 1.1 THE GENUS CROTALARIA L. Crotalaria L. (Fabaceae) is one of the largest genera of Papilionoideae consisting of ca. 700 species (Roux et al., 2013). It is the largest genera of vascular plants in tropical Africa (Polhill 1982). The genus common name is “Rattlepod’or rattlebox and is derived from the fact that the seeds become loose in the pod as they mature, and make a rattling sound when the pod is shaken. The name derives from the Ancient Greek κρόταλον, (Crotalon) meaning "castanet", and is the same root as the name for the rattlesnakes (Crotalus). The genus is almost cosmopolitan in distribution across tropical and subtropical regions of the world (Lewis et al., 2005), with it centre of diversity in Africa and Madagascar (ca. 543 species) (Polhill 1968, Polphil 1982, Roux et al., 2013) and a secondary center in India (ca. 92 species) (Ansari 2008; Sibichen and Nampy 2007). The genus is also widely distributed across the southern hemisphere, extending into Asia and North America. There are ca. 51 species of Crotalaria in Nigeria (Hutchinson et al., 1958) commonly distributed in tropical and sub-tropical regions with ca. 12 species in northern Nigeria (Adelanwa et al., 2014). Odewo et al., (2015) in their study of ecological distribution of the crotalaria in Nigeria reported only 3 species of the genus in Katsina. Members of Crotalaria differ in habit ranging from small shrub to herbs and may be annual or perennial. They are easily recognised by their yellow, whitish to purplish or bluish coloured flowers. The leaves are simple or one to three foliate, alternate, lanceolate to obovate. The genus can also be recognised by a combination of the following five diagnostic characters: rostrate kee, inflated fruit, 5+5 highly dimorphic anther arrangement (five long, basifixed anthers alternating with five short, dorsifixed ones), trichomes present on the style and macrocyclic pyrrolizidine alkaloids (Baker, 1914; Polhill, 1968; Van Wyk, 2005) Some of the species within the genus are widely used in agriculture, production of commercial products while some have medicinal and nutritional value (Polhil, 1982; Van Wyk, 2005; Pandey et al., 2010). The African and Madagascan species were revised by (Polhill 1982), following the revision of (Bisby 1973) and (Bisby and Polhill 1973). Polhill (1982) published an updated infrageric classification system of the genus where he recognised eight sections and 12 sub sections. Later on, Ansari (2008) revised the Polhill classification to accommodate the Indian species. He (Ansari 2008) maintained the eight sections by Polhill, but modifies the subsections. Recently, Le Roux et al., (2013) carried out a global infrageneric classification for the genus based on molecular and morphological evidence. They proposed a new sectional classification for the entire genus recognising eleven sections, after the modification of some sections recognised by Polhill. They raised some sub section to sectional level and abandoned some subsection due to non monophyly. 1.2 HISTORY OF CLASSIFICATION Phylogenetics is a modern approach of classification of living organisms which is concerned with reconstructing evolutionary history and relationships. It is also concerned with the recovery of the history of speciation (Thain and Hickman, 1995). Molecular phylogenetic techniques use DNA or amino acid sequences to build phylogenetic trees of species which shows the evolutionary relationship. The analysis is carried out using the following methods: Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI). Historically, classification systems were predominantly based on observation of specimen in the field or herbaria (e.g. de Candolle, 1838; Bentham and Hooker 1862-1883). The main source of data for these classification systems were micro and macromorphology. However, some taxonomist uses anatomical features, embryology and chemistry. In the early 20th century, this approach came to be regarded as unscientific, as the resulting classification system were not objective in any way, because two different taxonomist looking at the same plant group often arrived at two different classification system, because of differences of opinion with regard to which characters are useful for classification (Sokal and Sneath, 1963). Researchers made attempt to tackle this issue by developing numerical taxonomy also known as morphomeric analysis in the beginning of 1940’s. The idea was to look at all the morphology of the taxa and add many different morphological characters both the qualitative and quantitative to a data matrix and then classify the taxa based on overall similarity (Gilmour, 1940). Numerical taxonomy, however, have some problems more especially when it was used to look at relationship between taxa at, and above species level. Not all morphological characters can indicate or show relationship because some of them are homologous characters. Henning (1950; 1966) attempted to overcome the challenges by proposing his own method of phylogeny reconstruction, phylogenetic systematic, which is now known as cladistic. He realised that homologous characters cannot be used to infer evolutionary relationship and that only shared, advance characters (synapomorphies) could be used to infer phylogeny. He suggested that the principle of Occam’s razor analyses should be applied in analyses of characters, so as to find the simplest solution that explained the data. Henning is also aware that some characters appear the same through convergent evolution as a result of homoplasy, rather than because of an evolutionary relationship (Henning, 1950). This characters cause’s confusion in maximum parsimony analysis to date (Felsenstein, 1978). Academic debate known as the ‘cladist wars’ began as a result of Henning first publication between those who proposed cladistics and those who believed numerical taxonomy was a suitable way to infer evolutionary relationship (Ebach et al., 2008). The cladists won, and the numerical methods are now generally used to investigate relationships below the species level, rather than to resolve species relationships. Before the advent of DNA sequencing, cladistic method is the one use in classification and is mainly based on morphological characters, but it is more accurate and useful to reconstruct evolutionary relationships from DNA data. In using morphology, it is very difficult to be sure whether a character is truly synapomorphic or not, as identifying this involves decisions as to which morphological character states are primitive and which are advanced. This causes a serious debate among taxonomist (Sokal and Sneath, 1963). DNA data solved this problem as there was no need to decide subjectively which character states were primitive and which were advanced. 1.3 MORPHOMETRICS Morphometric analysis involves the multivariate analysis of a set of quantitative and qualitative morphological characters of individual specimens of the taxa of interest (sometimes referred to as operational taxonomic units, or OTUs). This is often used to determine whether closely related species have discrete or overlapping morphologies, which may be important in the taxonomic revision of a group. It is also used to ascertain the useful characters that can be used in classifying taxa of interest. The analysis, attempts to classify organism based on morphological similarity. It can be used to describe the pattern of similarities among taxa by ordination or cluster analysis (James and McCulloch 1990). Several angiosperm taxa have been reclassified using this method (El-Gazzar, 2008). Hussaini and Iwo (1992) work extensively on the genus Crotalaria and reported the genus phonological information which is based on conventional taxonomic methods. 1.4 INTERNAL TRANSCRIBE SPACER (ITS) NUCLEAR RIBOSOMAL DNA The internal transcribed spacer region of (ITS) of ribosomal DNA (rDNA) has been used successfully to achieve phylogenetic resolution at around the species level in many angiosperm genera (Hillis & Dixon, 1991; Baldwin, 1992; Baldwin et al., 1995). ITS is found in the part of the nuclear genome coding for ribosomal RNA. The RNA genes are made up of repeat units, each consisting of an IGS (inter-genomic spacer),18S gene, ITS1 spacer, 5.8S gene, ITS2 spacer and 26S gene. Within the IGS are two regions, the NTS (non-transcribed spacer) and ETS (external transcribed spacer). The structure of ITS is illustrated in Fig. 1.1. Figure 1.1: rDNA ITS structure (Linder et al., 2000) 1.5 STATEMENT OF THE PROBLEM There have never been taxonomic studies of the Crotalaria species in the study area, as such the current distribution, abundance and nomenclature of the species is not known. Similarly, majority of the species bear the same name on most of the herbarium collections deposited in the most largest herbarium having the bulk collections of the species in the region due to lack of proper identification while some have never been describe. These are impediments especially to the users of biodiversity and conservation agencies. The phylogenetic relationship and biography of Crotalaria species is not known since there has never been a molecular phylogenetic study on Crotalaria species in the study area. Plants in the genus Crotalaria are very important in bioremediation, ethnomedicine and agriculture globally. Ethnobotanical uses of the species of Crotalaria have never been documented in the study area. Since several species were found to be medicinal in other parts of the world e.g. Crotalaria is used in the treatment of diabetics (Pullaiah and Chandrasukha, 2003), skin infection, snake bite and stomach ache prevention (Verdhana, 2008), and there is tendency that species in the study area might be having some medicinal applications. The need to ascertain the species of Crotalaria that may serve these and many other purposes cannot be overemphasized. Numerical approaches have been very less utilized for taxonomic purposes in Crotalaria, even though there seems to be ample scope for their applicability for an assessment of the taxonomic relatedness of the genus. 1.6 JUSTIFICATION Through this research, it is hoped that the phylogenetic studies of the genus Crotalaria will help in establishing the evolutionary relationship that exist among all the species of Crotalaria, particularly those in the study area. It is also hoped that a monograph that will help in fully resolving all the taxonomic issues on the genus Crotalaria will be set, their correct nomenclature and also their diversity. Evaluation of the ethnobotanical uses of the species of this genus will also help in understanding there medicinal potentials to enable the people in the study area to make use of the plant in treating various ailments. Generally, the study will contribute to understanding the biodiversity and distribution of species of the genus Crotalaria in the study area and will immensely contribute to the knowledge of legumes biology particularly those endemic to the study area. 1.7 AIM AND OBJECTIVES The principal aims of the research in this thesis were to identify the potential members of the genus Crotalaria in Katsina and investigate the relationship between them. The secondary aim of the research was to conduct multidisciplinary studies (systematics and ethnobotany) on the genus Crotalaria. The study has the following objectives: Objective 1: To determine the phylogenetic relationship among the Crotalaria species found in the study area so as to establish their evolutionary relationship and to know their phylogenetic position in the current infrageneric system of classification of the genus Objective 2: To revise the taxonomy and nomenclature of the Crotalaria species found in the study area. Objective 3: To identify and document Crotalaria species with ethnobotanical applications in order to bring out their potential health benefit CHAPTER TWO LITERATURE REVIEW 2.1 TAXONOMIC HISTORY OF THE FAMILY FABACEAE The family Fabaceae taxonomically comprises of three subfamilies which includes Ceasalpiniodeae, Mimosoideae and Papilionoideae. The family is considered to be more closely related to Connaraceae and Sapindaceae based on anatomy, morphology and biogeographical distributions (Polphil and Raven 1981). The emergence of the three subfamilies is based on the floral parts which include the size and symmetry of the flower, arrangement of petals in the flower bud, having united or free sepals, number of stamens, presence of pleugram, embryo radicle shape and presence of rood nodules (Lewis et al., 2005). Based on the differences in the above mentioned characteristics, it is believed that Mimosoideae and Papilionoideae are unique distinct lineage in the family which arose independently within a paraphyletic basal caesalpiniod assemblage (Polhill, 1994). Before the advent of family-wide molecular phylogenetic studies, Polhill (1994) in his last formal classification recognized 39 tribes, 670 genera and 16, 850 species within the family. After intensive research of more than 10 years in molecular phylogenetics studies of the family, the tribal and generic classification of the family has been updated which recognizes 36 tribes, 727 genera and 19, 327 species (Lewiz et al., 2005). The genera with 500 or more species within the family includes (Acacia, Astragalus, Crotalaria and Indigofera) and about 40 genera have 100 species or more, the family also contain nearly 500 genera that contain up to 10 species (Lewis et al., 2005). There has been disagreement on whether the family Fabaceae should be treated as one (composed of Ceasalpiniodeae, Mimosoideae and Papilionoideae) or each sub family should be treated as individual family, evidence from morphology and molecules support the legumes being one monophyletic family. This view has been supported by recent molecular phylogenetic studies (Doyle et al., 2000; Kajita et al., 2001; Wojciechowski, 2003; Wojciechowski et al., 2004) showing strong support for a monophyletic group that is more closely related to Polygalaceae, Surianaceae and Quillajaceae, which together form the order Fabales (Sensu Angiosperm Phylogeny Group, 2003) 2.2 TAXONOMIC HISTORY OF THE TRIBE CROTALARIEAE The tribe Crotalarieae (Benth.) Hutch. is a member of the sub family Papilionoideae (Fabaceae) (Lewis et al., 2005). Members of Crotalarieae are monophyletic believed to have evolved from the tribe Liperieae (Goldblatt (981, Boatwright et al., 2008). It is the largest tribe within the paipilionoid legumes in Africa and also within the genistoid alliance, comprising about 51% of the genistoid legumes (Lewis et al., 2005). The reason why the tribe is large is because the genus Crotalaria which is a member in the tribe contains ca. 690 species (Polhill 1982). The tribe belongs to the core genistoids (Crisp et al., 2000) and currently comprises of 11 genera and ca. 1204 species (Van Wyk 2005) and ca 83% occurs in African continent and the other four genera (Aspalathus L., . Thunb., Rafnia Thunb. And Wiborgia Thunb.) occurs in the Cape Floristic Region (Van Wyk, 1991; Boatwright et al., 2008). The members of the tribe occur largely in Africa, with some of the species like Crotalaria, Lotonis and Rothia occurring on other continents. Aspalathu, Rafnia and Wiborgia are endemic to the Cape Floristic Region, while the genus Lebeckia is widely distributed throughout the cape and extends to some parts of Namibia (Boatwright et al., 2008). Polhill (1976) excluded the tribe Crotalarieae from Genistae (Adans.) Benth. s.l., following an in depth study of Geniseae He excluded the tribe based on the presence of a stamina tube that is open along the upper side (forming a closed tube in the Genisteae s.s.). Anarthrophyllum Benth., Dichilus DC., Melolobium Eckl. and Zeyh. and Sellocharis Taub. were also included in the Crotalarieae, but (Van Wyk and Schutte, 1995) moved back the genera to their previous tribe Genisteae. Results from morphological, chemotaxonomic studies (Van Wyk and Schutte, 1995) and recently molecular systematic studies (Boatwright et al., 2008a, 2009b, 2011) lead to the change in the generic delimitations within the tribe. Some lineage are raised to generic status and some there is also some nomenclatural changes (Boatwright et al.,2008a, 2011). Currently 16 genera in three valid clades are recognized. The Cape clade comprises of seven genera which includes Aspalathus, Wiborgia, Wiborgiella Boatwr. and B.-E. van Wyk, Calobota Eckl. and Zeyh., Lebeckia, Rafnia and Ezoloba B.-E. van Wyk and Boatwr. The Lotonis clade comprises of six general: Lotonis (D.C) Eckl. and Zeyh., Leobordea Del., Listia E. Mey., Pearsonia Dummer, Robynsiophyton R. Wilczek and Rothia Pers The Crotalaria clade comprises of only three genera: Euchlora Eckl. and Zeyh., Crotalaria L., and Bolusia Benth. Figure 2. 1. A cladogram showing the relationships of the three clades and 16 genera from an analysis of combined morphological and molecular evidence (Boatwright et al., 2008a and Le Roux et al., 2013) Some of the species within the tribe are of important commercially such as Aspalathus linearis, which is used for the production of rooibos tea (Van Wyk et al., 1997), and Lotonis bainesii Bak., is used as important fodder for animals (Bryan 1961). While some of the species within genus Crotalaria and Lotononis have been reported to have medicinal properties (Van Wyk 2005) and a few species are in traditional medicine in Lesotho to cure or ease a broken heart (Moteetee and Van Wyk 2007), other species from the same genera are toxic (WHO, 1988) 2.3 TAXONOMIC HISTORY OF THE GENUS CROTALARIA Carolus Linnaeus was the first to described the genus Crotalaria, he named 13 species in his Species Plantarum of 1753 which are Crotalaria perfoliata L., C. sagitalis L., C. juncea L., C. triflora L., C. villosa L., C. verrucosa L., C. lotifolia L., C. lunaris L., C. laburnifolia L., C. micans L., C. albanand L. C. quinquefolia L. (Linnaeus 1753). The number of the species within the genus increased to 37 (Lamarck, 1786). De Candolle (1825) reported 137 species. Thereafter the numbers increased to the total of 700 species that is accounted for at present (Le Roux et al., 2013). The genus is listed as one of the fifty largest seed plant genera (Mabberley, 2008). Detailed review of African species was given by (Baker, 1914), He revised and described 309 species in the continent. The most extensive study in the history of Crotalaria taxonomy was by (Polhill 1982), he conducted a thorough taxonomic revision on species in Africa and Madagascar where he reported 511 species. In Africa Thunberg (1823), reported 11 species from the Cape, South Africa. Similarly Harvey (1862) described 24 new species from the same region, which are now seen in other part of Africa (Le Roux et al., 2013). 21 species are enumerated from region of Empire of Ethiopia which comprises of Southern Egypt, Eastern Sudan, Yemen and Western Saudi Arabia by (Richard 1847). Verdoorn (1928) published a taxonomic revision of the genus where he recognized and reported 124 species in Southern Africa and South Tropical Africa, excluding Angola. Baker (1876) in his work on the genus in Tropical Africa, he treated 106 species. 24 species were collected in East Africa by Hildebrand and their description was published by (Vatke 1879). 24 years later, Tauber (1895) increases West African species to 56 in his publication. Milne-Redhead (1961) and polhil (1968, 1971a) conduct an in depth studies and revised the species within the genus in East Tropical Africa and reported 199 species in the area, 56 species from the same region were also published by (Taulbert 1895). In Angola 62 new species were described by (Wilczek, 1953a), he also revised 189 species from the same country (Wilczek, 1953b). Hepper (1958) revised the species of West Africa and reported 51 species and Da Torre (1960) described 36 new species and revised 138 species (1962) from the region, 26 species were treated in Namibia. In Ethiopia 85 species were treated (Thulin, 1983) similarly he reported enumerated 39 species in Somalia (Thulin, 1989). Du Puy and Labat (2002) conducted a research on Madagascar species and enumerated 53 species. Roy and Sharon (2005) described and illustrated new species of the genus Crotalaria mwangulangoi from the Udzungwq Mountains, Tanzania. Two new species of Crotalaria, C. cupricola Leteinturier and C. serpentinicola Leteinturier are reported from metallifeorus sites in Zimbabwe by (Leteinturier and Polhill 2003). A new species known as Crotalaria arrecta Hemp and Polhill was reported from Kenya, the species was previously confused with C. rhizoclada Polhill, the new species was described and illustrated by (Andreas and Polhill 2009). Odewo et al., (2015) reported 36 species in Nigeria in their studies on ecological distribution of the genus Crotalaria in Nigeria. Several literatures have been reported from Asia continent. Wight and Walker-Arnott (1834) conducted taxonomic revision of Indian species and reported 58 species in the country. Roxburgh (1832) described 7 new species in his Flora Indica. Descriptions of 77 species in British India were published by (Baker, 1876). 15 species are listed in the Red Data Book of Indian Plants (Nayar and Sastry 1987). According to (Ansari, 2008; Sibichen and Nampy, 2007) Crotalaria is the largest Fabacea genus in India with 92 species. De Munk (1962) in his revision of Malaysian legume, he published a list of 38 species within the genus. Adema (2006) in his work on taxonomic treatment for Malaysian species, he published some notes on the species that are endemic in the country. Wu et al., (2003) reported 6 species in their study on herbarium records, actual distribution, and critical attributes of invasive plants: genus Crotalaria in Taiwan. A detailed description of 42 species that occurs in china was reported by (Jianqiang et al., 2010). Lee (1979) investigate the species endemic to Australia and reported 7 species, while extensive studies on the native species was conducted by (Holland, 2002) and he reported 36 species in region. Important literature from Western Hemisphere includes: revision of the new world species by (Humbold, Bonpland and kunth) where they described nine new species. 32 species in Brazil were treated by (Bentham 1859); Andreia and Ana (2005) described and illustrated new species from Southeastern Brazil; Senn (1939) revised 31 species from North America and found that majority of the species are endemic to Mexico and West Indies. 12 unifoliate species were treated by (Windler 1974) from the same region; Windler and McLaughlin (1980) reported 11 species in the Panama region; nine newly species were reported by (Windler and Skinner, 1981) in America, they also described seven nomenclatural changes on the existing species.. Crotalaria avonensis DeLaney and Wunderlin was described for the first time as a new species from the xeric white sand scrub habitat of Highlands County, Florida by (Kris and Richard 1989). Lindsay and Michael (2012) reported 7 species in Alabama, in their studies: the genus Crotalaria (Fabaceae) in Alabama. The genus Crotalaria together with its sisters Bolusia Benth. and Euchlora Eckl. and Zeyh (100% BS, PP 1.0; Boatwright and al., 2008a) belongs to the tribe crotalarieae. The genus have the following characters which include a rostrate keel, highly inflated fruit, a hairy style, a 5+5 anther configuration, paired callosities on the standard petal and the presence of macrocyclic pyrrolizidine alkaloids (Polhill, 1982, Van Wyk and Verdoorn, 1990; Van Wyk, 2005). The closely sister to crotalaria which is the genus Bolusia differs from Crotalaria by having a helically coiled keel with a single callosity restricted to the standard petal blade (Van Wyk et al., 2010; Le Roux and Van Wyk, 2012). While with the other sister Euchlora differs from Crotalaria by lacking standard petal callosities and also has an obtuse to somewhat rostrate keel beak (Le Roux and Van Wyk, 2012). 2.4 INFRAGENERIC CLASSIFICATION The first infrageneric classification of the species within the genus was given by (Lamarck 1786), he divided the genus into two groups which are simple leaved group and trifoliate, digitate leaved group. Wight and Walker-Arnott (1834) used reproductive characters and reported 13 sub divisions with the two groups reported by Lamarck. Bentham (1843) used leaf shape and divided the genus into two groups which are simple leaved and trifoliate leaved, he further divided the simple leaved into seven sections and trifoliate leaved into 11 sections, this classification is similar to (Wight and Walker-Arnott 1834). Harvey (1862) maintained the simple leaved group and divides the species within the trifoliate group into Oliganthe and Racemosae, based on number of flower per raceme, thereby dividing the genus into three sections. Baker (1876) followed Harvey system of classification and adopts it but used fruit shape, flower arrangement and excluded Racemosae and create four additional groups (Chrysocalycinae, Sphaerocarpae, Oocarpae and Cylindrocarpae). Four sections were created based on leaf character (Simplicifoliae, Unifoliolatae, Trifoliolatae and Multifoliolatae) by (Taubert 1893) following Bentham’s classification. He also used vegetative and reproductive morphological characters to further divide the Simplicifoliae into seven series and Trifoliolatae into ten series. Baker (1914) adopted previous sections Simplicifoliae, Sphaerocarpae and Chrysocalycinae reported by (Baker 1876) and created three additional new sections and five subsections, the sections are Spinosae, Farctae and Eucrotalaria and the subsections are (Grandifloraei, Mediocriflorae, Oliganthae, Parviflorae and Stipulasae). Harms (1915), Senn (1939) and Peltier (1959) adopted the previous system of classification which was based on leaf, but Harms (1917) modified the section Chrysocalycinae, He removed C. niginas from the section and placed it in the newly created section Tetralobocalyx Harms. Wilczek (1953b) is not satisfied with using leaf as a tool for sectional classification because some species have both unifoliolate and trifoliolate leaves, therefore he disagreed with previous classification systems. He erected seven groups using petiole length, presence or absence of stipules and their size, which he named the groups “Group I-VII”. Polhill (1968) is not satisfied with any of the classification systems, and he also reported the difficulty of infrageneric classification of the species within the genus. He gave an account of the history and development of the classification systems for African Species (Polhill, 1968), He used a combination of flower morphology, legume and bracteole shape and divided the genus into 11 sections and seven sub sections. Bisby (1970, 1973) included 273 species and 52 characters that are similar to those used by Polhill (1968) which include floral characters, habit and stipule characters and conducted taximetric analyses. His findings were similar to that of Polhill’s (1968) classification system. Data from Polhill (1968) and Bisby (1973) were combined for the infrageneric classification system and found some discrepancies that were re-evaluates (Bisby and Polhill, 1973). Their finding resulted to an improved classification system (Bisby and Polhill, 1973; Polhill, 1982) which comprised of eight sections and nine subsections. The sections are: Grandiflorae, Chrysocalycinae, Incanae, Stipulosae, Hedriocarpae, Geniculatae, Schizostigma, Calycinae, and Crotalaria. Ansari (2008) used modified infrageneric classification system of (Wight and Walker-Arnott, 1834; Bentham, 1843 and Baker 1876) which are based on leaf and revised the taxonomy of Indian species. In his publication Ansari (2002) he reported nine sections and six subsections; which are not formally described. He considered Polhill’s classification system and updated his previous system of 2002 using the same sections as Polhill (1982), but listed only sections that are in India and included six sections and 12 subsections (Ansari, 2006, 2008). In his work he recognized four subsection within section Calycinae and four subsection within the section Crotalaria Le Roux et al., (2013) proposed sectional classification system for the entire genus for the first time based on morphological and morphometric studies and phylogenetic approach. Her new system comprises of eleven sections: Amphitrichae, Calycinae, Crotalaria, Geniculatae, Glaucae, Grandiflorae, Hedriocarpae, Incanae, Schizostigma, Borealigeniculatae and Stipulosae. She modified Geniculatae, Calycinae and Crotalaria sections. The subsections Stipulosae, Glaucae and Incanae are raised to sectional level, while some groups previously recognized as subsections are abandoned due to non-monophyly (subsections Chrysocalycinae, Hedriocarpae, Macrostachyae and Tetralobocalyx). Two new sections are recognized, Amphitrichae and Borealigeniculatae Table 2.1: Sectional and subsectional classisication for Crotalaria by Polhill (1968, 1982), Bisby and Polhill (1973), and Ansari (2008) Section/subsection Polhill (1968) Bisby and Polhill (1973), Polhill (1982) Ansari (2008) Section Grandiflorae + + + Section Incanae + - - Section Incanae Subsec Incanae + - - Section Incanae subsection Stipulosae + - - Section Incanae subsection Glaucae + - - Section Chrysocalycinae + + + Section Chrysocalycinae subsection Incanae - + + Section Chrysocalycinae subsection Stipulosae - + + Section Chrysocalycinae subsection Glaucae Section Chrysocalycinae subsection Chrysocalycinae - + - Section Chrysocalycinae subsection Tetralobocalyx + - - Section Purpureae + - - Section Hedriocarpae + + + Section Hedriocarpae subsection Priotropis + - - Section Hedriocarpae subsection Hedriocarpae + + + Section Hedriocarpae Subsection Macrostachyae - + + Section Macrostachyae + - - Section Geniculatae + + - Section Schizostigma + + - Section Calycinae + + + Section Calycinae subsection Alatae - - + Section Calycinae subsection Calycinae - - + Section Calycinae subsection Diffusae - - + Section Calycinae subsection Heylandiae - - + Section Crotalaria + + + Section Crotalaria subsection Bracteatae - - + Section Crotalaria subsection Crotalaria + + + Section Crotalaria subsection Longirostres + + + Section Crotalaria subsection Polyphyllae - - + Section Dispermae + + + 11 sections were reported by Polhill (1968) later Bisby and Polhill (1973) merged them to eight, Ansari (2008) reported six sections in India (Shweta and Arun, 2013).  Table 2.2: The infrageneric classification system from the crotalaria since 1973 (Le Roux et al., 2013) Bisby and Polhill (1973), Polhill (1982) Ansari (2006, 2008) Le Roux et al., (2013) Section 1. Grandilorae (Baker f.) Polhill Section 2. Chrysocalycinae (Benth.) Baker f. Subsect.: Chrysocalycinae Subsect.: Glaucae (Benth.) Bisby and Polhill Subsect.: Incanae (Benth.) Bisby and Polhill Subsect.: Stipulosae (Baker f..) Bisby and Polhill Subsect.: Tetralobocalyx (Harms.) Bisby and Polhill Section 3.: Hedriovarpae Wight and Polhill Subsect. Hedriocarpae Subsect. Macrostachyae (Benth.) Bisby and Polhill Section 4.: Geniculatae Polhill Section 5. Schizostigma Polhill Section 6.: Calycinae Wight and Arn Section 7.: Crotalaria Subsect.: Crotalaria Subsect.: Longirostres (Benth.) Polhill Section 8: Dispermae Wight and Arn Sect. 1:Calycinae Wight and Arn Subsect.: Alatae (Wight and Arn) A.A Ansari Subsect. Calycinae Subsect. Diffusae (Wight and Arn) A.A Ansari Subsect.Heylandiae A.A Ansari Sect. 2: Chrysocalycinae (Benth.) Baker f. Subsect. Incanae (Benth.) Bisby& Polhill Subsect. Stipolosae (Benth f.) Bisby& Polhill Sec. 3: Crotalaria Subsect. Longirostrae (Benth.) Polhill Subsec. Polyphyllae (Wight&Arn) A.A Ansari Sect.4: Dispermae Wight&Arn Sect.5: Grandiforae (Baker f.) Polhill Sec. 6: Hedriocarpae Wight&Arn. Subsect. Hedriocarpae Subsect. Macrostachyae (Benth.) Bisby&Polhill Sect.1: Hedriocarpae Wight&Arn. Emend. M.M. le Roux&B.-E. Van Wyk Sect.2: Incanae (Benth.) Polhill Sect.3: Schizostigma Polhill Sect.4: Calycinae Wight&Arn. Emend. M.M le Roux&B.-E. Van Wyk Sect.5: Borealigeniculatae Emend. M.M le Roux&B.-E. Van Wyk Sect.6: Crotalaria Emend. Sect.7: Stipulosae (Baker F.) . M.M le Roux&B.-E. Van Wyk Sect.8: Glaucae (Benth.) . M.M le Roux&B.-E. Van Wyk Sect.9: Geniculatae Polhill Sect. 10: Amphitrichae . M.M le Roux&B.-E. Van Wyk Sect.11: Grandiflorae (Baker f.) Polhill  2.5 ETHNOBOTANY Harshberger (1896) defined Ethnobotany as “the study of relationship between human being and vegetation in their environment, including medicinal uses”. Though the term “ethnobotany” was not coined until 1895 by the US botanist John William Harshberger, the history of the field begins long before that. In 77 AD, the Greek surgeon Dioscorides published “De Materia Medica”, which was a catalog of about 600 plants in the Mediterranean focusing on the economic potential of plants. It also includes information on how the Greeks used the plants, especially for medicinal purposes. In 1542, Leonhart Fuchs, a Renaissance artist, led the way back into the field. His “De Historia Stripium” cataloged 400 plants native to Germany and Austria. The 19th century witnessed the peak of botanical exploration. Alexander von Humboldt collected data from the new world and the famous Captain Cook brought back information on plants from the South Pacific. At this time, major botanicals gardens were started, for instance the Royal Botanical Gardens, Kew. Edward Palmer collected artifacts and botanicals specimens from peoples in the North American West (Great Basin) and Mexico from the 1860’s to the 1890s. Once enough data existed, the field of “aboriginal botany” was founded. Aboriginal botany is the study of all forms of the vegetables world which aboriginals people use for food, medicine, textile and ornamentals etc. The first individual to study the perspective of the plant world was Leopold Gleuck; a German physician working in Sarajevo at the end of 19th century. His published work on traditional medical uses of plants done by rural people in Bosnia (1896) has to be considered the first modern ethnobotanical work. Beginning in the 20th century, the field of ethnobotany experienced a shift from the raw compilation of data to greater methodological and conceptual reorientation. This is also the beginning of academic ethnobotany. The founding father of this discipline is Richard Evans Schultes. Plants used in traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases. A vast knowledge of how to use the plants against various illness may be expected to be accumulated in areas where the use of medicinal plants is still of great importance (Diallo, 1999). According to the world health organisation (WHO, 2003) more than 80% of the world population relies on traditional medicine for their primary health care Rural communities, in particular, depend on plant resources mainly for herbal medicine, food, forage, construction of dwellings, making household implement, fuel and shade. The use of medicinal plants as traditional medicine is well known in rural areas of many developing countries. Traditional healers claim that their medicine is cheaper and more effective than modern medicine (Veeramuthu et al., 2006). The indigenous traditional knowledge of medicinal plants of various communities where it has been transmitted orally for centuries is fast disappearing due to the advent of modern technology and transformation of traditional culture. Moreover, traditional healers are also decreasing in number and the younger generation are not interested in carrying on with the tradition and in most cases the knowledge has not been recorded, as such there is great danger that this cultural heritage and basis for future research may be lost forever. Therefore it becomes the responsibility of the scientific community to unravel this information and to document it for availability to the whole world for the benefit of human beings (Rajadurai, 2004). The Hausa land like Kano and Katsina are not left behind in the use of ethno medicinal plants where various groups of herbalist or consultant use different varieties of plants in the treatment of physical, mental, and social diseases. The healers may be sedentary or mobile, the latter following a regular circuit within a periodic market or moving randomly from village to village (Jinju, 1990). Indigenous knowledge has had a role to play in the understanding of the coexistence of man with fauna and flora over the centuries. Inspite of the great importance of this biological diversity and the amount of attention that is currently being given to traditional medicine at both national and international levels, many countries, particularly in Africa still do not have sufficient data on what biological resources they have, their location and how they may be utilized. Many of these biological diversities are used in local traditional medicine and have been reputed, through experience inherited from one generation to the other, to have useful medicinal activity. More than 80% of the world’s population is estimated to depend primarily on traditional medicine for the treatment of ailments (Cunningham, 1993). As noted by Okafor and Ham (1999), dependence on indigenous plants as medicine is due to unavailability of western medicine or high cost of it. It should also be noted that new medicinal compounds are often derived from species that have been useful as folk or traditional or native medicines for centuries, such as anti-hypertensive drug from Rauwolfia serpertina, anticancer from Catharantus roseus and antimalarial (Artemisinin) from Artemisia annua. Several reviews on approaches for selecting plants as candidate for drug discovery have been published (Phillipson and Anderson, 1989; Vlietnick and Vanden, 1991; Harvey, 2000; Farnsworth, 1988). Information from ethno botanical survey and subsequently phytochemical screening of such plants have been used in the past and are currently being pursued especially in developing countries as a means to initiate drug discovery efforts (Farnsworth, 1966). According to a survey of scientific literature, isolation of chemical compounds was stimulated by ethno botanical claims and a total number of 122 compounds were identified; 80% of these compounds were used for the same or related ethno medical purposes. These compounds were derived from 94 plants species (Farnsworth et. al, 1985). The problem being encountered currently in Nigeria and other developing countries is that, the people that hold this indigenous knowledge on the uses of plants are the older generations and the traditional healers. However, these custodians are decreasing in number due to death and other unforeseen occurrences. The younger generations have little interest in the practice due to urbanization and technological advancement. Hence, there is a danger that this knowledge will eventually disappear if nothing is done. Secrecy, superstition and lack of adequate records on the use of herbal medicine have also led to loss of invaluable heritage in herbal medicine. Treatment of mental illness is one of such area where documentation of how it is being treated is important, because it is an ailment that is hard to diagnose due to their subjectivity. Kankara et al., (2015) conducted ethnobotanical survey to document medicinal plants used for traditional maternal healthcare in Katsina state and found that members of family fabaceae are the most used plants in traditional medicine. Similarly Samaila and Munier (2015) reported that members of the family fabaceae are widely used in traditional medicine in their study on ethnobotanical survey of edible plants sold in Katsina metropolis markets. El-Gani (2016) in his attempt to document information on the traditional medicinal plant that are used in Nigeria, he reported that 325 species belonging to 95 families were used by most of the people in Nigeria for the treatment of various diseases and fabaceae has the largest number of species (42). Danjuma et al., (2015) gave an account on medicinal plants in metropolitan Kano, Nigeria and reported 29 inventories trees of the area are used for various medicinal applications. Similarly Danjuma and Darda’u (2013) reported similar number of species in a study of medicinal plants used in Katsina state in Northwestern Nigeria.. After the time of Harshberger (1896) to the present date, several authors have tried to give a description of subject ethnobotany and its scope, methodology, its various disciplines sub-disciplined and potential etc. Schutles (1960) had written on tapping our heritage ethno-botanical. He had suggested three methods of ethnobotany among the primitive peoples. He also gave some examples of the plant used during ancient period. Jain (1964) wrote on the role of botanist in folk lore research. He writes that folklore research involves the study of all aspect of intellectual and material culture of indigenous or backward people. Jain (1965) outlined the prospects by some new or less known medicinal plants resources. Jain (1986) gave an overview of the subject ethnobotany, an indication of the significant research during last thirty year in this field and also showed how ethnobotany is an interdisciplinary science. Schutles (1986) tried to bring the attention of scientists to ethnobotanical conservation. For many years, he has been engaged on the studies in Pristline forest of the Amazon and other regions of tropical South America. Arora (1987) described ethnobotany and its role in the domestication and conservation of native plant genetic resources. He gave the detail account of this important area where ethno-botanists have still a great to do. 2.6 ECONOMIC IMPORTANCE OF CROTALARIA Crotalaria species are annual shrubs very useful in agriculture (Magingo, 1992), as green manure and cover plants in plantations. They are also used as a source of diet for livestock. Many of the species are known to be nodulated with soil Rhizobia (Allen and Allen 1981, Faria et al., 1989) and they are also good in fixing atmospheric nitrogen. Sustainable crop production is achieved through the management of soil fertility and cover crops play a key role in soil fertility through a reduction in synthetic nutrients applied. Legumes are effective in the fixation of nitrogen and can accumulate large amounts of biomass that help to increase the nutrient availability and organic matter in the soil. Some of the long term benefits obtained from the use of cover crops include weed suppression through competition or allelopathy and possible insect control (Phatak et al., 2002). While some species of Crotalaria are poisonous, others like C. retusa, C. micronata, C. falcata and C. vogelii remain some of the important fodder plants for cattle and small ruminants. (Nuhu et al., 2000) also reported that Crotalaria species are widely used in Zaria, Nigeria in feeding of sheep and cattle. Crotalaria species are widely used in veterinary pharmacy in preventing liver disease (Nwude and Ibrahim, 1980, Nuhu, 1999). In Tanzania Crotalaria comosa Bak provides nitrogen to the crops intercropped with and assist in the control of weeds and nematodes (Mukurasi, 1986). The species within the genus like Crotalaria recta L. are used as food source by larvae of Lepidoptera species, the insect also used the plant as defense against their predators (Thomas 2003). Cook and White (1996) revealed that C. retusa L. seeds as source of fibres, silage and green manure when removed from pods by pounding. According to Akintayo, (1997) oils derived from Crotalaria bongensis Bak, C. naragutensis Hutch and C. lachnophora Desu. Seeds are not suitable for use as edible oil and soap production but many however, are useful for the production of paint and shampoos. Crotalaria is also used in the treatment of diabetics (Pullaiah and Handrasekhar- Naidu, 2003), skin infection, snake bit and stomach ache prevention (Verdhana, 2008). CHAPTER THREE METHODOLOGY 3.1 STUDY AREA The study was conducted in Katsina state, northern Nigeria. Katsina state is located between latitudes 11°08′N and 13°22′N and longitudes 6°52′E and 9°20′E. The state covers an area of 23,938 sqkm. The state is bordered by Niger Republic to the North, Jigawa and Kano States to the East, Kaduna State to the South and Zamfara State to the West. The state has 34 Local Government Areas. Figure 3.1: Map of Katsina showing the study area 3.2 TAXON SAMPLING Plant materials were collected through field survey of the genus Crotalaria across the study area. The plants were identified based on morphological features seen in relevant literatures. Specimens from Herbarium of the department of Biological sciences, Ahmadu Bello University Zaria were also used as a guide in indentifying the plants. Plants were collected following the procedure for collection of plants specimen for standard herbarium (Natalie, 2009). A twig of about 24 cm was collected for all the encountered species and they were pressed immediately to maintain the morphological features of the plant to be use as voucher specimen. While collecting, young leaves of each species were collected in an envelope containing silica gel for molecular studies and each envelop is labeled accordingly. Bolusia was suggested to be the sister group of Crotalaria (Polhill, 1996, 1982), and this has been confirmed by molecular studies by Boatwright et al., (2008) with Bootstrap support 92% and PP 1.0. It was also reported that the Crotalaria – Bolusia clade is sister to monotypic genus Euchlora (99% BS; PP 1.0; Boatwright et al., 2008). Based on these findings both Bolusia and Euchlora were used as outgroups in this study. Other sequences of Crotalaria species not present in the study area were downloaded from Genbank in the interest of creating more complex phylogeny and to know the phylogenetic position of the species collected for this study in the current infrageneric classification. 3.3 MORPHOMETRIC ANALYSIS One of the objectives of this study is the identification, description and revision of the taxonomy of members of the genus Crotalaria present in the study area by using morphometric techniques. For this purpose, specimens were collected across the sahelian region of Katsina State through field observations and collections noting their vegetative and floral morphology. The specimens were pressed so as not to affect the leaves in terms of length and width and floral characters. In addition some specimens in the herbaria of Umaru Musa Yaradua University, Katsina were used in the study. The total number of 49 individuals of the 8 species found were used in the morphometric analyses. Collecting details of the taxa included in the analysis are given in table 3.1 Table 3.1: List of species within the genus Crotalaria taken for the study S/No Name of taxa Collector Number Locality 1. Crotalaria macrocalyx Yaradua SSY1 Jibia 2. C. macrocalyx Muhammad MM1 Jibia 3. C. Senegalensis Yaradua SSY2 Kaita 4 C. Senegalensis Yaradua SSY3 Kaita 5 C. Senegalensis Yaradua SSY4 Kaita 6 C. Senegalensis Yaradua SSY5 Mashi 7 C. Senegalensis Yaradua SSY6 Mashi 8 C. Senegalensis Yaradua SSY7 Mashi 9 C. Senegalensis Yaradua SSY8 Mani 10 C. Senegalensis Yaradua SSY9 Mani 11 C. Senegalensis Yaradua SSY10 Daura 12 C. Senegalensis Yaradua SSY11 Daura 13 C. sp. Yaradua SSY12 Sandamu 14 C. sp. Yaradua SSY13 Sandamu 15 C. sp. Yaradua SSY14 Kaita 16 C. sp. Yaradua SSY15 Kaita 17 C. sp. Yaradua SSY16 Kaita 18 C. sp. Yaradua SSY17 Mashi 19 C. sp. Yaradua SSY18 Mashi 20 C. sp. Yaradua SSY19 Jibia 21 C. sp. Yaradua SSY20 Jibia 22 C. sp. Yaradua SSY21 Jibia 23 C. sp. Mustapha SSY22 Mani 24 C. sp. Yaradua SSY23 Jibia 25 C. sp. Mustapha SSY24 Jibia 26 C. sp. Yaradua SSY25 Jibia 27 C. sp. Yaradua SSY26 Jibia 28 C. sp. Yaradua SSY27 Jibia 29 C. retusa Yaradua and Bello Bello395 Kaita 30 C. pallida Yaradua SSY28 Jibia 31 C. pallida Yaradua and Bello Bello396 Jibia 32 C. pallida Yaradua and Bello Bello397 Daura 33 C. pallida Yaradua and Bello Bello398 Daura 34 C. pallida Yaradua and Bello Bello399 Daura 35 C. pallida Yaradua and Bello Bello340 Daura 36 C. pallida Bello Bello341 Mani 37 C. pallida Bello Bello342 Mani 38 C. pallida Bello Bello343 Jibia 39 C. pallida Yaradua SSY28 Kaita 40 C. pallida var. obovata Yaradua and Bello Bello344 Kaita 41 C. pallida var. obovata Yaradua and Bello Bello345 Kaita 42 C. pallida var. obovata Bello Bello346 Kaita 43 C. pallida var. obovata Yaradua and Bello Bello347 Kaita 44 C. pallida var. obovata Yaradua SSY29 Kaita 45 C. pallida var. obovata Yaradua and Bello Bello348 Jibia 46 C. pallida var. obovata Yaradua and Bello Bello349 Jibia 47 C. pallida var. obovata Yaradua and Bello Bello350 Daura 48 C. pallida var. obovata Yaradua and Bello Bello351 Daura 49 C. pallida var. obovata Yaradua and Bello Bello352 Daura A characters set consisting of 21 characters for morphometric analysis of Crotalaria was adapted from a character set used previously by Le Roux et al. (2013) and Britto et al.(2011) with some slight changes. Out of the 20 characters 15 were quantitative and 6 were qualitative. The morphometric character set used is shown in table 3.2. Table 3.2: List of qualitative and quantitative characters and character states used in morphometric analysis. S/No Character States Quantitative characters 1 Number of leaflet (Nl) mm 2 Length of petiole (Lp) mm 3 Length of leaflet (Ll) mm 4 Width of leaflet (Wl) mm 5 Length of fruit (Lf) mm 6 Length of seed (Ls) mm 7 Length of pedicel (Lp) mm 8 Number of flower per axial 9 Length of calyx (Lc) mm 10 Number of seeds 11 Length of standard petal (Lsp) mm 12 Width of standard petal (Wsp) mm 13 Length of wing petal (Lwp) mm 14 Width of wing petal (Wwp) mm 15 Length of keel petal (Lkp) mm Qualitative characters 16 Habit (H) Herb (0), small shrub (1) 17 Life form (lf) Annual (0), Perennial (1) 18 Presence of hair (Ph) Presence (0), Absent (1) 19 Shape of leaflet (Sl) Lanceolate (0), Spatulate (1), Ethptic (2), Elliptic (3), Cuneate (3), Obovate (4) 20 Inflorescence position (Ip) Terminal (0) Axial (1) 21 Pods size (Ps) Slightly exceeding calyx (0), far exceeding calyx (1) 3.3.1 Data Analysis Multivariate analyses were carried out by cluster analysis (CA), principal coordinates analysis (PCA) and jaccards similarity coefficient using PAST 3 program. The cluster analysis will show whether the species are distinct species or not while the principal coordinates analysis will show that characters that are usefull in showing delimination among the species. The jaccards similarity coefficient was calculated to know how similar the species are. All the 21 morphological characters including both quantitative and qualitative for all the 49 specimens, were used for the analysis and each individual specimen was considered as an operational taxonomic unit (OUT). Jaccards similarity coefficient was also determine usinf In all the analysis, the data was first log10 transformed for standardization of the data matrix. CA was used to show the similarities among the species, while PCA was used to shows the character that are useful in showing the similarities and differences across the species within the genus. 3.4 MOLECULAR STUDY 3.4.1 DNA Extraction EasyprepTM Plant Genomic DNA Kit was used for DNA extraction. The Kit was design for efficient recovery of genomic DNA up to 60kb in size from fresh and dried plant tissue. DNA was extracted from all the specimens as outlined in the user manual of the kit. Silica gel dried leaf material was ground to powder using a pestle and 50 mg of powder dry tissue was put in 2.0 ml tube then 600 µl Buffer PL1 and 10µl of beta mercaptoethanol were added and the tube was vortex to mix vigorously to disperse all clumps in Deluxe Mixer (American Scientific Products Division of American Hospital Supply Corporation McGraw ParkTQ IL 60085). The samples were in incubated at 65oC for 10 minutes in a dry bath (Barns Stead/Thermolyne 2555 Kerper Boulevard DUBUQUE, IOWA 52001 USA model no: DB 17015, serial no: 821980600241). The samples were mixed twice during the incubation by inverting the tubes and vortexing, after vortexing 200 µl Buffer PL2 was added to the tube and it was mixed by vortexing for 10 seconds followed by incubation on ice for 5 minutes, and centrifuged at 11, 000 rpm for 10 minutes in centrifuging machine (Centrifuge 5415C, eppendorf), 400 µl of the supernatant was transfered to a clean 1.5 ml eppendorf tube, 400 µl of Isopropanol was added to the tube and the tube was mixed well by vortexing for 5 seconds, centrifuged at 12, 000 rpm for 2 minutes to precipitate the DNA (this remove polysaccharides and improve DNA binding ability to the spin column). The supernatant were carefully discarded by using pipette to draw the supernatant to avoid dislodging the DNA pellet. The tube was inverted on absorbance paper towel for 1 minute to drain any residual ethanol (this is also done carefully taking care of the DNA pellet), 300 µl of pre heated ddH2O (65oC) was added to the tube containing the DNA pellet and it was vortex for 10 seconds to mix the DNA well, it was then briefly incubated at 65oC to help dissolve the DNA, 150 µl of Buffer PL3 and 300 µl of 100% ethanol were added to the tube containing the mixture of DNA pellet and pre heated ddH2O (65oC), the tube was mixed well by vortexing for 5 seconds (a precipitation was formed but it doesn’t interfere with the DNA binding to column). The samples were transferred to a clean column and centrifuged at 11, 000 rpm for 1 minute. The flow was discarded through and the column was put back to the collection tube. This was repeated once. The column was centrifuged at 11, 000 rpm for 1 minute. The column was transferred into a clean 1.5 ml tube, 100 µl of pre warmed (65oC) elution buffer was added and the tube was centrifuged at 11, 000 rpm for 1 minute to elute DNA (smaller volumes will significantly increase DNA concentration but give lower yield, use of more than 200 µl of buffer for elution is not recommended). The eluted DNA was put back to the column for second elution to yield another 20-30% of the DNA band. 3.4.2 Polymerase Chain Reaction (PCR) The mixture used in the PCR was the same for all the specimens. For each specimen, 14 µl of ultra pure water, 1.5 µl of both the forward and reverse primers, 3 µl of DNA sample were used, giving a total of 20 µl per reaction. Reactions were run on an MJ Research PTC – 200 Peltier Thermal Cycler. The primer used to amplify the ITS region is given in table 3.1, together with their sequences. Table 3.3: Primers used in PCR reactions Region to be amplified Primer name and Direction Primer sequence ITS (Nuclear) ITS5 (forward) 5’-GGA AGT AAA AGT CGT AAC AAG G-3’ (White et al., 1990 ITS (Nuclear) ITS4 (reverse) 5’-TCC TCC GCT TAT TGA TAT GC -3’ (White et al., 1990) ITS region was chosen because result from previous studies by Le Roux et al. (2013) have shown that it provide robust resolution with the genus under studies. Similarly Boatwright et al. (2008, 2011) reported that the region provides robust resolution at higher taxonomic levels for the tribe Crotalarieae. Chan et al. (2010) in their studies also reported the efficiency of using ITS region in species identification within the Fabacea family. The PCR process involved an initial denaturation phase of 5 minutes at 95oC by 35 cycles of 40 seconds at 94oC: 40 seconds at 50oC (annealing); 40 seconds at 72oC (extension); and a final extension phase of 5 minutes at 72oC. All PCR products were run out on agarose gel to check successful reactions, before being purified using Qiagen, QIAquick PCR purification kits, according to the protocols supplied by the manufacturer. 3.4.3 Agarose Gel Electrophoresis Agarose gels were made by mixing 1.5g agarose gel powder with 100 ml of 1 X TBE (Tris-HCl Borate EDTA) buffer, heating until dissolved and adding 25 µl of 1mg/ml ethidium bromide, once the solution had cooled sufficiently. The gel was then poured into a tray with an appropriately sized comb and left to set for about 30 minutes. DNA extracts were run on agarose gels to indicate approximate concentration, using 3 µl of extracted DNA mixed with 5 µl of loading solution. PCR products were also loaded onto gels in 3 µl quantities, mixed with 5 µl of loading solution. Size of PCR fragments was detected using a Bioline Hyperladder I, 1kb size ladder. Gels were run at approximately 80V in an electrophoresis tank for approximately 90 minutes, and visualised using UV transillumination. 3.4.4 Gel Extraction The DNA band was cut from the gel for gel extraction and it was put into clean 2.0 ml tube. Gel buffer was added to the tube and incubated for 10 minutes at 50oC to dissolve the Gel, it was mixed 3 times during the incubation. The dissolved gel with the DNA was put into DNA binding column, it was then centrifuged at 11, 000 rpm for 1 minute. The flow was discarded through and the column was put back to the collection tube, the process was repeated once, 750 µl of DNA wash buffer and ethanol were added to the column and centrifuged at 11, 000 rpm for 1 minute. The empty column was centrifuged at 13, 000 rpm for 4 minutes to remove residual ethanol. The column was transferred to a clean 1.5 microcentrifuge tube. 25 µl of ddH2O was added to the column and incubated at 50oC for 5 minutes and then centrifuged at 11, 000 rpm for 1 minute to elute the DNA. The column was discarded and the eluted DNA was stored in microcentrifuge tube at – 20oC for sequencing reactions. PCR product for sequencing: 5 µl of the DNA 5 µl of ddH2O 2 µl of riverse primer The product was used for the sequencing reactions. The sequencing was done using ABI 3730 capillary DNA sequencer in DNA lab Kaduna. 3.5 PHYLOGENETIC ANALYSIS Resulting sequences from the sequencing reactions were assembled and edited using Staden package (Staden et al., 1998). Bioedit was used to align the sequences (Hall, 1999), ClustalW multiple alignment was used to aligned the sequences electronically followed by manual adjustments in the same ClustalW (Bello et al., 2015). All positions containing gaps and missing data were eliminated (Shweta et al., 2013). Phylogenetic analyses were done using Bayesian Inference Analysis MrBayes. 3.6 ETHNOBOTANICAL DATA The field methodological framework chosen for this study was that used in ethnobotany (Martin, 1995; Samaila and Munier 2015) and based on method given by Gidey (2010), semi-structured interviewees; observation and guided field walk with informants were employed to obtain ethnobotanical data. Field research was conducted by collecting ethnobotanical information during structured and semi-structured interviews with knowledgeable people native in each site territory. For each species recorded one questionnaire was filled. Even though, a structured questionnaire had to be filled direct questions were avoided. The basic information needed was taken during the conversation No special selection criteria were used in the choice of the informants because one of the aims of this work was to assess the breadth of popular heritage in the field of wild edible plants, knowledge which is widespread among locals. However, most of the interviewees were men and women between 40-60 years old. During oral interview the questions to the respondents are their names, occupation, knowledge of medicinal uses of the plants, part used and the use of the plants. The respondents cooperate in answering questions. The respondents response in filling the questionnaire given to them, the literate ones filled the questionnaire themselves while those that can’t fill the questionnaire I filled it for them while answering the questions, the questionnaire used in the study is in appendix 1. Data obtained were collated and arranged to give the botanical names, and the local names as well as their uses and the part(s) used. 3.6.1 Data analysis The results obtained during the survey were analyzed using the Relative Frequency of Citation (RFC) (Kankara et al., 2015). 3.6.1.1 Relative Frequency of Citation (RFC) This was used to calculate the importance of a particular species in treating ailments. It was determined using RFC = Fc/N where Fc is the number of respondents who cited a particular species and N is the total number of the respondents (Tardio and Pardo-de-Santayana, 2008). CHAPTER FOUR RESULTS AND DISCUSSION 4.1 RESULT 4.1.1 Morphometric Result 4.1.1.1 Clustering The result of the cluster analysis separated 7 clusters (at Euclidian distance of 0.2; figure 4.1). The cophenetic correlation coefficient value of r = 0.964 obtained in the analysis indicates a very good fit between the triangular distance matrix and the phenogram (Sneath and Sokal 1973, Rohfl, 1998). The groups were recognized as distinct taxa at different taxonomic hierarchies if all their OTUs did not mix between clusters. All the a priori groups formed distinct clusters with the exception of G (Crotalaria pallida) and H (Crotalaria pallida var. obovata) whose specimens intermixed and formed a large cluster, this is because they are the same species H is variety of G. Figure 4.1. Unweighted Pair Group Method with Arithmetic mean (UPGMA) phenogram resulting from cluster analysis. Cophenetic correlation (r) = 0.964. Table 4.1: Similarity matrix based on Jaccard’s coefficient   A B C D E F G H A 1 0.933333 1 1 1 0.933333 1 1 B 0.933333 1 0.933333 0.933333 0.933333 0.866667 0.933333 0.933333 C 1 0.933333 1 1 1 0.933333 1 1 D 1 0.933333 1 1 1 0.933333 1 1 E 1 0.933333 1 1 1 0.933333 1 1 F 0.933333 0.866667 0.933333 0.933333 0.933333 1 0.933333 0.933333 G 1 0.933333 1 1 1 0.933333 1 1 H 1 0.933333 1 1 1 0.933333 1 1 The result of the Jaccard’s similarity coefficient varied between 0.867 and 0.993,indicating closer relationships between the species. 4.1.1.2 Ordination The ordination analysis based on the result of PCA separated the 49 specimens into 7 groups corresponding largely to those obtained in the cluster analysis (figure 4.2) Principal component 1 accounted for 72.3% of the variation while principal component 2 accounted for 9.2% of the variation. The loading of the PC 1 and 2 are presented in table 4.1. The character mostly correlated with the first PCA axis with value (r > 0.50) is: length of petiole 0.77, while the character correlated with the second PCA axis with value (r > 0.50) is: number of flower per axis 0.87 Figure 4.2: Plot of the first two principal component analyses (PCA) obtained from analysis of the morphological data set of specimens of the species within the genus Crotalaria. The first and second PCA axes explain 72.3% and 9.2% of the total variation between all the taxa, respectively. The result of the PCA showed that majority of the clusters in the ordination plot, corresponds largely to those obtained by cluster analysis. The priori groups identified in cluster analysis (Euclidian distance 0.2, figure 1) were supported by the ordination analysis. Legend: A = Crotalaria macrocalyx, B = Crotalaria sp., C = Crotalaria senegalensis, D = Crotalaria sp., E = Crotalaria retusa, F= Crotalaria sp., G= Crotalaria pallida, H= Crotalaria pallida var. obovata. Figure 4.3: PCA loading of the characters. It showed the PCA loading of the contribution of each character in similarities within the species. Length of petiole, length of leaflet have higher loading in the result. Table 4.2: Loadings of the first and second components in the principal components analysis Axis Eigenvalue % variance 1 0.352441 72.337 2 0.0450442 9.2451 3 0.0368382 7.5608 4 0.0254375 5.2209 5 0.0179612 3.6864 6 0.0048452 0.99445 7 0.0012719 0.26105 8 0.0011449 0.23499 9 0.0007317 0.15018 10 0.0005689 0.11676 11 0.0003361 0.068992 12 0.0002474 0.050785 13 0.000187 0.03839 14 0.000128 0.026278 15 4.00E-05 0.0082159 4.1.2 Species Identification During the field survey 7 different species were found, five of them were identified using the application of numerical taxonomy while the other three could not be identified. The identified species are C. retusa, C. senegalensis, C. pallida, C. pallida var. obovata and C. macrocalyx. Herbarium specimen and young leaf to be used for molecular studies were successfully collected from the study area. On the diversity of the species, the 2 of the unidentified species are not common. Details of the collections are presented in Table 4.3. Table 4.3: Collections of Crotalaria Taxon Collection number Locality information Leaf collected C. macrocalyx SSY 1 Jibia Y C. senegalensis SSY2 Mashi Y C. retusa Bello 395 Kaita Y C. pallida SSY 28 Dankama Y C. pallida var. obovata SSY 29 Daura Y C. sp. SSY 12 Mashi Y C. sp. SSY 15 Jibia Y C. sp. SSY 18 Dankama Y 4.1.3 DNA Extraction, Gel Electrophoresis DNA of all the 7 species encountered was successfully extracted, the genomic DNA was used to amplified the ITS region of each species using the ITS primers ITS4 and ITS5. The bands from the result of the gel electrophoresis are around 680bp. The result of the gel electrophoresis shown on plate 1. Plate 4.1: Result of gel electrophoresis of the 7 species. 4.1.4 Phylogenetic Analysis Phylogenetic relationships were determined using Bayesian inference on MRBAYES run on CIPRES portal. The data set included 58 accessions (43 species including two out groups). Out of the 43 species, 36 represent all infrageneric groups across all the continents while the remaining seven are the collections from the present study area. The analysis showed that Crotalaria is monophyletic (PP, 1.0). In the tree some clades are strongly supported (PP value above 8.0) while other clades are mederately supported (PP value lessthan 8.0). The results showed that four (C. macrocalyx, C. retusa, C.sp., and C. pallida) of the seven species sampled were distributed across the various sections of Crotalaria (Hediriocarpae clade, Calycinae clade, and Stipulosae clade respectively), while three, Crotalaria sp. Crotalaria sp. and Crotalaria sp. were distributed across (Incanae clade, Longirostres clade respectively). However, more sequences from other species in the C. senegalensis complex coupled with morphological studies will reveal if C. senegalensis complex should be raised to a sectional status or lumped in Longirostres clade. Our results also showed that C. retusa is resolved as sister to southern African C. papilosa in Calycinae clade. The positions of the studied samples across all the sections are strongly supported (PP 1.0), but relationships between the clades and species relationship differs slightly. Relationship between Longirostres and Calycinae is strongly supported with (PP 1.0), this shows that two of the studied samples (Crotalaria retusa and Crotalaria sp.) are closely related and the relationship between Crotalaria retusa and C. pilosa is moderately supported (PP, 0.70). Incanae and Hediriocarpae relationship is also well supported (PP, 0.99) and relationship between South African Crotalaria pallida and West African Crotalaria pallida is also strongly supported (PP, 0.89). Relationships between Crotalaria sp. and Crotalaria burkeana in the Incanae section is strongly supported (PP, 1.00). While the relationships between Crotalaria nataliata and Crotalaria sp. in the Stipolosae section is weakly supported (PP, 0.63). Figure 4.4 Phylogenetic relationships of Crotalaria (43 taxa) based on Bayesian Inference analysis of ITS region. A= Crotalaria macrocalyx, B= C. sp. C= C. senegalensis, D= C. pallida E= C. retusa, F=C. sp., G=C.sp. 4.1.5 Taxonomic Description and Revision Plate 4.2: vegetative and flower morphology of C. macrocalyx Crotalaria macrocalyx Benth. London Journal of Botany 2: 572 (1843) Type: Heudelot M., 205 (P, holotype) Shrub, annual, leaves 3- foliate; lanceolate, leaflet length 42mm, leaflet width 10mm length of petiole 3mm, inflorescence terminal with 6 to 8 flowers, length of calyx 11mm, length of standard petal 8mm, width of standard petal 8mm, length of wing petal 10mm, width of wing petal 5mm, length of fruit 8mm, pods slightly exceeding calyx, number of seed 2, length of seed 3mm, length of pedicel 3mm, hair present. Plate 4.3: Vegetative, flower and fruit morphology of C.sp. Crotalaria sp. Shrub, annual, leaves 3- foliate; spatulate, leaflet length 30mm, leaflet width 6mm, length of petiole 4mm, inflorescence terminal with 7 to 8 flowers, length of calyx 10mm, length of standard petal 8mm, width of standard petal 8mm, length of wing petal 7mm, width of wing petal 5mm, length of fruit 8mm, pods slightly exceeding calyx, number of seed 9, length of seed 1mm, length of pedicel 2mm, hair present Plate 4.4: Vegetative, fruit and flower morphology of C. senegalensis Crotalaria senegalensis (Pers.) Bacle ex DC. Prodromus Systematis Naturalis Regni Vegetabilis 2: 133 (1825) Type: Adanson 24 (MPU isotype). Shrub, annual, leaves 3- foliate; Ethptic, leaflet length 33mm, leaflet width 12mm, length of petiole 20mm, inflorescence terminal or axial with many flowers, length of calyx 5mm, , length of standard petal 12mm, width of standard petal 9mm, length of wing petal 7mm, width of wing petal 7mm, length of fruit 11mm, pods far exceeding calyx, number of seed 10, length of seed 2mm, length of pedicel 4mm, hair presence. Plate 4.5: Vegetative, flower and fruit morphology of C.spp Crotalaria sp. Shrub, annual, leaves 3- foliate; elliptic, leaflet length 40mm, leaflet width 17mm Length of petiole 40mm, inflorescence terminal or axial many flower, length of calyx 6mm, , length of standard petal 8mm, width of standard petal 10mm, length of wing petal 12mm, width of wing petal 7mm, length of fruit 15mm, pods far exceeding calyx, number of seed 10, length of seed 3mm, length of pedicel 3mm, hair presence. Plate 4.6: Vegetative, flower and morphology fruit of C.sp. Crotalaria sp. Shrub, annual, leaves 3- foliate; Ethptic, leaflet length 30mm, leaflet width 13mm, length of petiole 30mm, Inflorescence terminal with 1 to 3 flower, length of calyx 4mm, length of standard petal 8mm, width of standard petal 7mm, length of wing petal 6mm, width of wing petal 3m, length of fruit 16mm, pods size far exceeding than calyx, number of seed 10, length of seed 2mm, length of pedicel 3mm, hair presence. Plate 4.7: Vegetative, flower and fruit morphology of C. pallida var. obovata Crotalaria pallida var. obovata (G. Don) Polhill Kew Bull, 22:265 (1968) Type: Polhill (BM holotype) Shrub, annual, leaves 3- foliate; obovate, leaflet length 40mm, leaflet width 25mm, length of petiole 40mm, inflorescence terminal or axial with many flowers, length of calyx 4mm, length of standard petal 9mm, width of standard petal 9mm, length of wing petal 9mm, width of wing petal 7mm, length of fruit 20mm, pods far exceeding calyx, number of seed 12, length of seed 2mm, length of pedicel 2mm, hair presence. Figure 4.8: Vegetative, flower and fruit morphology of C. pallida Crotalaria pallida Aiton Hortus Kewensis 3:20-21 (1789) Type: James Bruce s.n (BM, holotype) Shrub, annual, leaves 3- foliate; Ethptic, leaflet length 40mm, leaflet width 25mm, length of petiole 40mm, inflorescence terminal or axial with many flowers, length of calyx 4mm, length of standard petal 9mm, width of standard petal 9mm, length of wing petal 9mm, width of wing petal 7mm, length of fruit 20mm, pods far exceeding calyx, number of seed 12, length of seed 2mm, length of pedicel 2mm, hair presence. Figure 4.7: Vegetative morphology of C. retusa Crotalaria retusa L. Species Plantarum 2:715 (1753) Type: Herman 84 (BM, lectotype) Shrub, perennial, leaves 3- foliate; cuneate, leaflet length 50mm, leaflet width 12mm, length of petiole 3mm, inflorescence terminal with many flowers, length of calyx 8mm, length of standard petal 12mm, width of standard petal 9mm, length of wing petal 12mm, width of wing petal 5mm length of fruit 33mm, pods far exceeding calyx, number of seed 14, length of seed 2mm, length of pedicel 6mm, hair absent. 4.1.6 Ethnobotany The species within the genus were found to be used in treating various ailments in the study area and they are very effective in the treatment. The name of each species, part used, ailment treated are given in the table below. Majority of the respondent having the knowledge of the medicinal uses of the plant are usually old age people and they are herbalist.  Table 4.4: List of medicinally utilised species Name of species Local name Disease Part used Preparation Route RFC Crotalaria Pallida Farar Biya rana Skin infection Whole plant Decoction Body bath 0.41 Eczma Whole plant Decoction Dermal Urinary problems Leaves Decoction Oral Fever Whole plant Decoction Body bath Swelling of joint Root Poultice Dermal Tumor Seed Maceration Oral Stomachache/indigestion Root Maceration Oral Ulcer Leaves Powder Oral Orchitis Whole plant Maceration Oral Hypertension Whole plant Maceration Oral Epileptic Whole plant Decoction Body bath Crotalaria retusa Hemoptysis Roots Maceration Oral 0.29 Lung diseases Leaves Maceration Oral Scabies Leaves Maceration Oral Fever Leaves Decoction Body bath Impetigo Leaves Decoction Body bath Cold Flower/leaves Decoction Oral Scorpion sting Seeds Direct Oral Chicken pox Leaves Ointment Dermal Crotalaria sp. Gyadar awaki Wound Leaves/seeds Ointment Dermal 0.07 Ulcer Seeds Direct with honey Oral Crotalaria macrocalyx Katsemi Dendruf Whole plant Powder Dermal 0.07 Back pain Whole plant Powder Dermal Crotalaria senegalensis Kahwar tankarki Fever Whole plant Decoction Oral 0.07 Gonorrhea Whole plant Powder Dermal Crotalaria Pallida var. obovata Bakar biya rana Dizziness Leaves Powder/Decoction Dermal/Oral 0.07 Orchitis Whole plant Maceration Oral  4.2 Discussion Many researchers worked on the genus Crotalaria globally across the whole continents, new species were described by several authors, while some revised previously described species and attempting to establish a workable infrageneric classification (De Lamarck, 1786; Wight and Walker-Arnot, 1834; Bentham, 1843; Baker, 1871; Harms, 1915; Polhill, 1968, 1982; Bisby, 1973; Ansari, 2006, 2008; Le Roux et al., 2013) among others. Members of Crotalaria found during field work and included in this study are Crotalaria senegalensis, C. retusa, C. pallida, C. macrocalyx, C. pallida var. obovata and three unidentified species totaling 7 species. Odewo et al., (2015) in their study on ecological distribution of the genus Crotalaria in Nigeria reported only three species in Katsina which are Crotalaria naragutensis, Crotalaria atrorubens. However, C. atrorubens is not reported in this study; species morhologically resembling to Crotalaria naragutensis which is Crotalaria pallida is reported. Chromosomome counts and cytomorphological studies of Crotalaria from Northern Nigeria conducted by Adelanwa et al., (2014) reported nine species and none was reported from the study area. One of the unidentified species is regarded as Crotalaria ononoides (Odewo et al., 2015)the species is not Crotalaria ononoides because after our morphometric analysis, it was discovered that the morphological features of the species are not the same with type specimen of Crotalaria ononoides. For example, the leaf shape of the species is spatulate, while that of Crotalaria ononoides is lanceolate to elliptic or obovate. The species’s 3- foliate leaves are unequal in size while that of Crotalaria ononoides are equal in size. The size of the fruit of the species is 8mm while that of Crotalaria ononoides is 12mm among others. Britto et al., (2011) reported that phenetics proves its robustness in identifying the closer species instead of relying on few vegetative characters which poses a great confusion in identifying species. One of accepted infrageneric classification system of the genus Crotalaria was based on morphometric (Bisby 1973, Bisby and Polhill 1973). Morphological characters both vegetative and floral have been used for constructing classifications (Agyeno et al., 2014a). Similarly, Jayeola (2001) reported the efficiency of utilizing vegetative and floral parts in numerical evaluation of similarities among taxa. Agyeno et al., (2014b) reported that morphology of leaf, habit and life span played a very important role in delimiting members of the genus Crotalaria due to their discontinuity or discreteness. Our findings agreed with his findings because leaf morphology is the character that showed great variation among our sampled species. The finding of Raj et al., (2011) also highlighted that qualitative characters such as habit, leaf type and quantitative characters such as pod length and seed number, petiole are phylogenetically important in the genus. Our findings also prove that the characters are effective in showing similarities among the species within the genus. In this study we consider characters such as petiole length which were not reported earlier and the character was found also to be effective in morphometric analysis of the genus. Britto et al., (2011) in their study on Identification of agronomically valuable species of Crotalaria based on phenetics, conducted numerical analyses of 58 morphological characters of 12 accessions belonging to the genus and reported that habit, leaf, pods shape, pods size, seed number are important characters showing All the infrageneric classifications before (Le Roux et al., 2013) were based on morphology. Polhill (1968) used reproductive characters, Bisby (1970, 1973) employed morphometric in his classification, later on, Polhill and Bisby combined their findings and published a final system in 1973 (Bisby and Polhill, 1973). Polhill in 1982 while working on African and Madagascan species reported eight sections which are currently in used. Ansari while working on Indian species reported the positions of Indian species where he modified Polhill, (1982) infrageneric classification system (Ansari, 2006, 2008), his work was also based on morphology. Le Roux et al., (2013) proposed global infrageneric classification system for the first time where they combined morphological and molecular data. They reported 15 sections. This system of classification is the one currently in used. Species from West Africa included in this studywere not captured in Le Roux et al 2013 infrageneric classification but the result of our analysis supported the system of system of classification, our sample species were distributed across Longirostres, Calycinae, Incanae, Hidiriocarpae and Stipolosae clades respectively with moderately to strong posterior probability value in Bayesian Inference analysis (PP, 8.0 to 1.0). In addition the studies species shared some morphological features with their sister species in the clade they belong. The species were reported to be effective in treating various ailments. Nuhu et al., (2009) reported the effective use of the species within the genus Crotalaria in treating various ailments in northern Nigeria. The used of Crotalaria pallida in treating skin infection such eczema is also reported by (Greenand et al., 1987, Marini, 1959). Our findings that Crotalaria retusa is used to treat chicken pox is also reported by (Powell, 1976, Holdsworth, 1977). Lochman (1987) also reported the use of the plant to heal scorpion sting which was also reported here. Crotalaria pallida is reported to have high medicinal value with RFC= 4.1, while Crotalaria retusa have RFC = 0.29. Crotalaria macrocalyx, Crotalaria senegalensis and one of the unidentified species have RFC = 0.07 respectively and this is the first time of reporting their medicinal uses. CHAPTER FIVE SUMMARY, CONCLUSION AND RECOMMENDATIONS 5.1 SUMMARY Crotalaria L. (Fabaceae) is one of the largest genera of Papilionoideae which includes ca. 700 species. The genus is cosmopolitan in distribution across tropical and subtroptical regions of the world; it has its main centre of species diversity in Africa and Madagascar and secondary radiation in the southern hemisphere. The principal aim of the research is to identify the potential members of the genus Crotalaria in Katsina, investigate the evolutionary relationship among them and determine their phylogenetic positions in the current system of classification. Another important objective of the research is to document ethnobotanical potentials of the species. All these were achieved in the research. Seven different species and one variety were reported in the dissertation, which were identified through morphometric and using online catalogue. Out of the seven species only four were identified which are Crotalaria retusa, Crotalaria senegalensis, Crotalaria macrocalyx and Crotalaria pallida the variety is identified as Crotalaria pallida var. obovata while the other three are not identified. The result of the morphometric in cluster analysis showed that all the groups were recognized as distinct taxa at Euclidian distance of 0.2 (cophenetic correlation value (r) 0.964). The result showed that Principal component 1 and 2 accounted for the variation in the ordination with 72.3 and 9.2% respectively and also the results also showed that leaf morphology is a good character that shows variation across the species within the genus.The species in the study area are revised and taxonomic description of each species is reported describing both the vegetative and floral parts. To determine the phylogenetic relationship and phylogenetic position of the species, DNA of each species was extracted and subjected to PCR to amplify the ITS region using ITS4 and ITS5 primers. Sequencing reaction was perform using the PCR product after clean up. The sequences were assembled and aligned. The data were analysed using bayesian inference analysis run using MrBayes. The result showed that the genus is monophyletic (PP, 1.00). The results showed that four (C. macrocalyx, C. retusa, C.sp. and C. pallida) of the seven species sampled were distributed across the various sections of Crotalaria (Hediriocarpae clade, Calycinae clade, and Stipulosae clade respectively), while three, Crotalaria sp,. Crotalaria sp. and Crotalaria sp. were distributed across (Incanae clade, Longirostres clade respectively). Relationship between Longirostres and Calycinae is strongly supported with (PP 1.0), this shows that two of the studies samples Crotalaria retusa and Crotalaria sp. are closely related. Crotalaria macrocalyx and Crotalaria senegalensis are also related though they are not in the same section. Ethnomedicinal uses of the species is also reported in the dissertation, the species reported have great ethnomedicinal uses in the treatment of ailments Crotalaria pallida is reported to have high medicinal value among the species treating about 12 ailments The aim and objectives of this this dissertation is achieved and the findings contributed a lot by evaluating and providing the phylogenetic relationship between members of the genus and determining their phylogeneic position in the current infrageneric classification system which was never reported before. The research also revealed that the species regarded as Crotalaria ononoides by previous researchers is identified wrongly. Also the ethnomedicinal uses of some of the species are reported for the first time globally and for all the species is documented for the first time in the study area. Therefore this thesis contribute to science be providing new knowledge on the taxonomy and ethnobony of the genus Crotalaria in Katsina. 5.2 CONCLUSION The study reported seven different species and one variety for the first time in the study area and also it is the first molecular systematic studies of the genus Crotalaria in West Africa and second in the whole African continent. The result supportted the monophyly of the genus Crotalaria and agreed with the current infrageneric system of classification of the genus. Three of the seven species were unidentified, they may be new species which the author will work on them and look at the possibilities of describing them as new species. 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Knowledge on medicinal plants Yes No Part of plant used Leaf Root Flower Stem Seed Whole Plant nut tuber Mode of Collection: Dry Fresh Season of plant cultivation: Rainy Season Dry Season Mode of preparation : Decoction Maceration Direct Body bath 21 17