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Assessment of the Use of DNA Barcoding for Identification of Some Cichlid

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Assessment of the Use of DNA Barcoding for Identification of Some Cichlid COPYRIGHT AND CITATION CONSIDERATIONS FOR THIS THESIS/ DISSERTATION o Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. o NonCommercial — You may not use the material for commercial purposes. o ShareAlike — If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original. How to cite this thesis Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujcontent.uj.ac.za/vital/access/manager/Index?site_name=Research%20Output (Accessed: Date). Assessment of the use of DNA barcoding for identification of some cichlid species By OLUSHOLA AHMED OLALEYE DISSERTATION Submitted in fulfilment of the requirements for the degree MASTER IN ZOOLOGY at the UNIVERSITY OF JOHANNESBURG May 2017 SUPERVISOR: PROF HERMAN VAN DER BANK CO-SUPERVISOR: PROF JOHN MAINA DECLARATION I declare that this dissertation hereby submitted to the University of Johannesburg for the degree MAGISTER SCIENTIAE (Zoology) has not been previously submitted by me for the degree at this or any other University, that it is my work in design and execution, and that everybody who contributed and all materials contained therein has been duly acknowledged. OLUSHOLA AHMED OLALEYE May 2017 Page | i ACKNOWLEDGEMENT My deepest gratitude goes to Prof. Herman van der Bank and Prof. John Maina for their supervision. Special thanks to Dr Olivier Maurin, Dr Kowiyou Yossofou and Mr Ronny Kabongo for their support and assistance in making the analyses of this study possible. Also, my sincere gratitude to Pavlinka for her immense and swift support in providing articles and textbooks that where difficult for me to get. My heartfelt appreciation to Prof. Mitchelle van der Bank and Faculty of Science University of Johannesburg for provision of funding for this project. A big thank you my dear colleagues and friends: Mariam Adeoba, Anifat Bello, Henry Ihenachio, Ojelade Solomon, Oloruntoba Bamigboye, Oluwatobi Bamigboye, Olakunle Oyekanmi, Akinseye Samuel, Lindi Steyn and Dr Bezeng S. Bezeng for their assistance, suggestions and corrections in the different parts of this study. I appreciatively acknowledge Dr Lolo Mokae, Dr Ololade Shonubi, Dr Seyi Olokede and Dr Tola Odubajo for their enormous support, advices and time throughout the study. I thank the African centre for DNA Barcoding for its infrastructural support for the duration of this project. Page | ii To my ever-supportive family, my dad Alhaji Azeez Olaleye, my mom Elizabeth Olaleye, my sister and her family the Ogunbinu-Peters and my brother and his family the Olaleyes; I gratefully appreciate and thank you for believing in me and always encouraging me in all my endeavors. Also to my lovely wife Palesa, my daughter Matshepo and son Olusegun for being my greatest motivation for accomplishing this study. I truly appreciate all your support, prayers and encouragement my wife. Lastly and above all appreciations, I thank God Almighty for bestowing strength, Mercies and grace through all the duration of this study. Oluwa mi e seun gan. Oluwa mi modupe. Page | iii ABSTRACT Species identification and delineation are very important in studies such as biodiversity, conservation biology, ecology and evolutionary biology. Cichlids are the common model group of fishes which are used to explicate the mechanisms of adaptation, speciation and diversification. Application of combined molecular genes (such as 16S, Cytochrome oxidase subunit I (COI), histone H3 & recombination activating gene two TMO4C4) on the phylogeny of cichlids has played an important role in cichlid phylogenetic studies. Here, identification of cichlids were assessed using DNA barcoding and phylogenetic tree reconstruction. All analyses were based on a single mitochondrial gene COI. Sequences of Alcolapia grahami and Thoracochromis albolabris were generated for the first time along with mined sequences from Barcode Of Life Data base systems (BOLDsystems). BRONX (Barcode Recognition Obtained with Nucleotide eXposés) was also used to confirm unique sequences representing each species. The performance of COI gene was tested based on three identification metrics namely best close match (bcm), near neighbour (nn) and BOLD identification criteria (BOLD id) using SPIDER in R. Next, occurrence of barcode gap was established on the difference of furthest intraspecific distance among its own species and the closest, non-conspecific distance (nonConDist – maxInDist). A maximum parsimony and a Bayesian inference (bi) tree were reconstructed to further investigate species delimitation among cichlid fishes. Both nn and bcm showed 89% true and 87% correct identification respectively in the dataset whereas BOLD id gave a lower correct identification (54%). There were well-supported values at each node of bi tree reconstruction, indicating evolutionary divergence among cichlids. COI gene performed well in the identification of Alcolapia and Thoracochromis (nn and bcm = 100%). It also performed well in the phylogenetic positions of Alcolapia and Thoracochromis (support values of 92% and 100% respectively). However, populations of Alcolapia grahami were not fully resolved by COI gene. The COI barcoding gene is viewed as an efficient tool for enhancing species identification and delimitation. Morphological data and nuclear genes or microsatellites should be added to resolve the ambiguity in the problematic specimens. Page | iv TABLE OF CONTENT Declaration…………………………………………………………………………..… i Acknowlwdegments…………………………………………………………………. ii Abstract………………………………………………………………………..………. iv List of figures…………………………………………………………………………. viii List of tables…………………………………………………………………………... ix 1. Introduction……………………………………………...………………..………… 1 1.1 General introduction to cichlids.………………………………………………… 1 1.2 Cichlids of economic importance……………………………………………....... 2 1.3 Distribution of cichlids……………………………………………………………... 3 1.4 Biogeography of cichlids………………………………………………………….. 6 1.5 Lacustrine Old World cichlids…………………..……………………….……….. 9 1.5.1 Brief biogeography of some Lake Tanganyika cichlids……………… 9 1.5.2 Brief biogeography of some Lake Malawi cichlids…………………… 10 1.5.3 Brief biogeography of some Lake Victoria cichlids…………………… 10 1.5.4 Brief biogeography of Lake Magadi cichlid …………………………… 11 1.6 Riverine Old World cichlids……….……………………………………………..... 13 1.6.1 River Congo cichlid distribution ………………………………………... 13 Page | v 1.6.2 Cichlid fishes of the southern African freshwater systems…………... 14 1.7 Phylogenetic relationships among cichlid assemblages………………………… 15 1.8 DNA barcoding – a molecular tool for species identification………………….… 18 1.9 Hypotheses and Objectives of the study………………………………………… 23 2. Materials and methods…...………………………………………………………….. 23 2.1 Locations and habitat of species sampled……………………………………….. 25 2.2 Sampling of taxa…………………………………………………………………… 25 2.3 DNA extraction……………………………………………………………………… 30 2.4 PCR amplification…………………………………………………………………… 32 2.5 PCR reaction clean-up……………………………………………………………… 33 2.6 DNA cycle sequencing……………………………………………………………… 34 2.7 DNA sequence analysis…………………………………………………………… 34 2.8 DNA Barcoding……………………………………………………………………… 34 2.9 Phylogenetic tree reconstruction…………………………………………………… 36 3. Results...……………………………………………………………………………….. 38 3.1 Extraction of DNA and amplification of COI gene……………………………….. 38 3.2 Sequence mining and filtering……………………………………………………… 40 3.3 DNA barcoding……………………………………………………………………… 40 3.4 Outline of nucleotide variation….……………………………………………….… 50 Page | vi 3.5 Phylogenetic relationship among Cichlidae…...………………………………… 51 3.6 Relationship among some species within the Oreochromini lineage.………… 57 4. Discussion…………………………………………………………...………………… 59 4.1 Phylogenetic relationships…………..……………………………………………… 61 5. Conclusion………………………………………………………...…………………… 66 References……………………………………………………………………………..… 68 Appendix………………………………………………………………………………….. 91 1. Cichlid authorities………………………………………………………………… 91 2. The distribution of cichlids used in this study..………………………………... 94 3. The position of base pair where variations occurred within the two populations of Alcolapia grahami sampled from Fish Springs Lagoon (FSL) and South West Lagoon (SWL)……………………………………………………………...100 Page | vii LIST OF FIGURES Figure 1. Distribution of the family Cichlidae showing some barcoded locations (squares) of cichlid species in the world (www.boldsystems.org) .......................... 5 Figure 2. Map of Africa indicating the distribution of some African cichlids ..…...... 27 Figure 3. Distribution of Magadi tilapia (Alcolapia grahami).………………..………. 28 Figure 4. Distribution of genus Thoracochromis in southern Africa ………………. 29 Figure 5: Gel electrophoresis products of clean DNA of two African cichlid fishes viewed in 1.5% agarose gel with the aid of ultra-violet light…………………….…... 38 Figure 6: Gel electrophoresis products of PCR products and clean PCR products…………………………………………………………………………..………. 39 Figure 7: Barcode gap evaluation of cichlid family represented in this dataset…… 46 Figure 8. Maximum parsimony tree based on mtDNA COI data of some cichlids………………………………………………………………………………….…. 52 Figure 9. One of the 73 most parsimonious trees based on mtDNA COI data of representative of Oreochromini lineage…………………………………………...….
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