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Addis Ababa University Addis Ababa, Ethiopia June 2020 PLANT DIVERSITY, ETHNOBOTANY AND BARCODING OF MEDICINAL AND COSMETIC PLANTS IN KALU AND BATI DISTRICTS OF AMHARA REGION, ETHIOPIA NURYA ABDURHMAN BIRHANU Addis Ababa University Addis Ababa, Ethiopia June 2020 PLANT DIVERSITY, ETHNOBOTANY AND BARCODING OF MEDICINAL AND COSMETIC PLANTS IN KALU AND BATI DISTRICTS OF AMHARA REGION, ETHIOPIA NURYA ABDURHMAN BIRHANU A Thesis Submitted to the Department of Plant Biology and Biodiversity Management Presented in Fulfilment of the Requirements for the Degree of Doctor of Philosophy (Biology: Plant Biology and Biodiversity Management) Addis Ababa University Addis Ababa, Ethiopia June 2020 ADDIS ABABA UNIVERSITY GRADUATE PROGRAMMES This is to certify that the thesis prepared by Nurya Abdurhman Birhanu, entitled: “Plant diversity, Ethnobotany and Barcoding of Medicinal and Cosmetic Plants in Kalu and Bati Districts of Amhara Region, Ethiopia” and submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy (Biology: Plant Biology and Biodiversity Management) complies with the regulations of the University and meets the accepted standards with respect to originality and quality. Signed by the Examining Board: Name Signature Date 1. ___________________ (Examiner) __________________ __________________ 2. __________________ (Examiner) __________________ __________________ 3. Prof. Zemede Asfaw (Advisor) __________________ __________________ 4. Prof. Sebsebe Demissew (Advisor) __________________ __________________ 5. Prof. Hugo De Boer (Advisor) __________________ __________________ __________________________________ _____________ Chair of Department or Graduate Programme Coordinator Abstract Plant diversity, Ethnobotany and Barcoding of Medicinal and Cosmetic Plants in Kalu and Bati Districts of Amhara Region, Ethiopia Nurya Abdurhman, PhD Dissertation Addis Ababa University, 2020 The study was conducted in Kalu and Bati Districts, South Wollo Zone of Amhara Region, Ethiopia. It was aimed at documenting plant diversity in selected forest patches, ethnobotany and barcoding of medicinal and cosmetic plants. Vegetation data were collected from 50 and 30 sampling plots (20 m × 20 m) for Anabe and Gerfa-ourene forest patches respectively using transect method. For shrubs and herbs 5 m x 5 m and 1 m x 1 m subplots were laid within each quadrat respectively. Ethnobotanical data were collected by administering semi-structured interviews with randomly sampled 300 informants, through direct field observation, 20 focus group discussion and in eight local markets. For DNA barcoding sequences, samples were collected both in voucher form and by silica gel and analyzed at the laboratory of Natural History Museum, University Of Oslo, Norway. Agglomerative hierarchical classification with the application of R-computer programing (R Version 3.0.2) was used to identify plant communities. Simple preference ranking, direct matrix ranking, informant consensus factor (ICF) and fidelity level (FL) were calculated to analyze the ethnobotanical data. Genomic DNA was extracted from silica-gel-dried leaves, voucher specimens and powdered medicinal plant samples to generate barcodes for ITS, rbcL and matK using specific primers and PCR amplification. All barcode sequences were queried using NCBI BLAST to cross-check morphological identifications. From Anabe and Gerfa-ourene forest patches a total of 128 plant species which belong to 114 genera and 60 families were recorded. From the total, 108 of the plant species representing 97 genera and 52 families were collected from Anabe Forest patch whereas 84 plant species representing 75 genera and 46 families were from Gerfa-ourene Forest patch. From the hierarchical cluster analysis, the vegetation in Anabe and Gerfa-ourene Forest patches were classified in to three plant community types each. The ethnobotanical study in Kalu District revealed 129 medicinal plant species that belong to 108 genera and 59 families. In Bati District, however, 94 traditional medicinal plant species belonging to 78 genera and 49 families were reported. The majority of human remedy preparations were from leaves (43% in Kalu and 29% in Bati) followed by roots (11% in kalu and 17% in Bati) and the highest ICF values were recorded for back pain in humans in both districts (0.94 in Kalu, 0.96 in Bati). The highest FL values of human medicinal plants were recorded for Withania somnifera (93.5%) in Kalu District, and Terminalia brownii (95.96%) in Bati District. About 30.34% of the medicinal plants in both districts were cited for women’s health problems and for herbal cosmetics.The amplified products were used for sequencing and 154 samples were sequenced for three barcode regions (ITS, rbcL and matK). BLAST was used to compare sample sequences to the reference database and sequencing success was high for rbcL (97.4%) and ITS (96.1%), but lower for matK (76.0%). Anabe and Gerfa-ourene Forest patches are rich in species composition having 128 plant species which belong to 114 genera and 60 families. Both districts are rich in their medicinal plant composition having 145 medicinal plant species that belong to 116 genera and 62 families. Kalu and Bati Districts are rich in “women’s plants” and herbs used as cosmetics (44 species, which belong to 39 genera and 31 families). DNA barcoding method was attempted for identifying 154 medicinal and cosmetics plant materials including from markets with high percentage of success. Key words: Bati, DNA barcoding, ethnobotany, ITS, Kalu, matK, rbcL. i Acknowledgments Many people have contributed a lot for the successful accomplishment of this dissertation. I cannot mention all in this part. However, I would like to take this opportunity to express my gratitude to my advisors Prof. Zemede Asfaw (AAU), Prof. Sebsebe Demissew (AAU) and Prof. Hugo De Boer (University of Oslo) for their unreserved support, guidance and motivation during my study. Their assistance throughout the thesis work, editing, revising and commenting on the manuscript and encouraging me all the time is invaluable! I would like also to pass heartfelt thanks to my supervisor the late Prof. Ensermu Kelbessa who had been support and encouraging me in my research work up to he passed away. My sincere thanks also go to Wollo University for providing an opportunity and for sponsoring my PhD study. I am also grateful to the Graduate Programs of Addis Ababa University for financing the necessary costs; NORPART project of Oslo University for funding expenses related to laboratory work, travel grant and research stay. I would also like to acknowledge my Department, Plant Biology and Biodiversity Management, especially Dr. Bikila Workneh (chair of the department) and Dr. Ermias Luelekal (stream advisor) for their incredible support and encouragement. I am also indebted to thank the people in Kalu and Bati districts who directly or indirectly participated in this research. Words cannot express how grateful I am to all of you for all of your support and assistance during my field work. The field assistants, particularly Ato Mohammed- nur Aba and Omar Shehu, and security guards Ato Seid Oumar, Muhidin Abidelah, Ato Mohammed Seid, Ato Seid Muhe and the administrative staff at several levels (Kebele, Wereda, Zone and Region) are also highly acknowledged. My sincere thanks also go to the technical staff members of the National Herbarium (ETH), Ato Melaku Wondafrash, W/ro Shewangiziw Lemma and Ato Wege Abebe for their collaboration in ii my herbarium work and their motivational support. The executive secretary of Plant Biology and Biodiversity Management, W/ro Yirgalem H/Selassie is also highly acknowledged, she has been a great help throughout my PhD work. The National Meteorological Services Agency of Ethiopia is also acknowledged for providing meteorological data of Kalu and Bati districts. Members of the DNA lab in the University of Oslo, especially, Birgitte Lisbeth and Audun Schrøder-Nielsen also acknowledged for their kind support in all aspects of my laboratory work. I am deeply indebted to Prof. Brita Stedje and Prof. Charlotte Sletten and their family for their unforgettable hospitality, support and great encouragement throughout my stay at University of Oslo. I would like to pass my gratitude to Dr. Samison Shemelis, Dr. Alemayehu Kefalew, Dr. Amare Kenaw, Ato Mamo Sisay, W/rt Lamirot Abera, Ato Tesfay Godifay, Ato Wondye kebede, Dr. Addise Yalew, W/ro Helen Moges, Ato Tesfay Assefa, Ato Ambaye Abayu and Dr. Birhan Alkadir for their valuable encouragement and moral support throughout my study. W/rt Lamirot Abera, W/ro Kedihja Mekonnin, W/ro Alemitu Lire and her family, W/ro Helen Moges and her family, Ato Solomon Boru and his family, W/ro Azimera Wolde, W/ro Yewubdar Abebaw, W/ro Habitam Getaneh and her family, Ato Hafte Amsalu, W/ro Dyma Mulleta and her family and W/ro Embet Belay are truly acknowledged for their kindest support, encouragement and prayers during my research work. Dr. Tamrat Bekele (AAU), is deeply acknowledged for his persistent encouragement during my research work and Prof Zerihun Woldu (AAU), is also heartily acknowledged for providing useful reference, and encouragement during my research period. I am very thankful to my friends and colleagues W/ro Kidist Bere Tareke and her family, Ato Solomon Yemane, Ato Yohannes Mekonnin and his family, Dr. Dessalegn Chala and his family and Dr. Abel Gizaw for their unreserved support and encouragement during my research stay at iii Oslo. I would also like to extend my gratitude to all my friends and colleagues at Addis Ababa and Wollo University for your friendly and professional support as well as for the good times we spent together. Dr. Moges Kibret as a new chair of our department at Wollo University and Mr. Belay Melese are sincerely acknowledged for their kind support. I am indebted to my beloved family, my mother “Gagay”, my sister W/ro Fatuma Abdurrahman (Fate), W/rt Lamirot Abera Ali, my brother Nurhussien Abdurrahman, my niece Zafran Abdulkadir (Mitye) and Ziyad Abdulkadir (Keabu), who have always been with me encouraging me to accomplish my PhD work. My sister W/ro Fatuma Abdurrahman is sincerely acknowledged for her moral and material support starting from my childhood up to now.
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