This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Ward, Sophie Title: Morphological and Molecular Approaches for Assessing Diversity of East African Freshwater Fishes General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. MORPHOLOGICAL AND MOLECULAR APPROACHES FOR ASSESSING DIVERSITY OF EAST AFRICAN FRESHWATER FISHES Sophie Ward A dissertation submitted to the University of Bristol in accordance with the requirements for award of the degree of Masters by Research in the Faculty of Science. July 2020 Student Declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University’s Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate’s own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. Signed: S.Ward Date: 31/07/20 Candidate Number: 37577 Supervisors: Martin Genner, University of Bristol. Christos Ioannou, University of Bristol. i THESIS ABSTRACT Conservation and management of freshwater fish in Africa can benefit from improved knowledge of the diversity of species present. This thesis focusses on methods to delimit species using morphological measurements and DNA barcodes. In Chapter 1, I discuss known levels of freshwater fish biodiversity in Africa, threats to African biodiversity, the existence and complications of cryptic species, the current knowledge surrounding freshwater fish biodiversity in Tanzania, catfish biodiversity, and the use of DNA barcoding data in systematics. In Chapter 2, I present a study investigating the use of molecular data in taxonomy, specifically DNA barcoding, in studying Tanzania’s freshwater fish biodiversity. Two different DNA markers (mitochondrial COI and 12S) were used to assess diversity among different barcoding genes and evaluate their utility in separating distinct taxa using neighbour-joining phylogenies. Both markers were equally successful in delimiting morphologically-identified specimens from the Ruaha-Rufiji-Kilombero river catchment into species groups. However, comparisons of these sequences to global reference sequence databases highlighted the absence of publically-available validated reference data for the species included in our study. In Chapter 3, I present a study investigating the use of linear morphological measurement in species identification, focussing on Chiloglanis catfish from South-Eastern Africa. The results support the use of linear measurement data in species delimitation, but also highlight the presence of genetically-distinct yet morphological similar “cryptic” species. Overall, the findings suggest morphological diversity within Chiloglanis has arisen from stabilising selection on the overall fluvial phenotype, potentially coupled with adaptation to specific characteristics of local river systems. In Chapter 4, I discuss the implications of these results for future freshwater ichthyofaunal research in Africa. ii Acknowledgements I would like to give my sincere thanks to many people for their support and involvement during my Masters by Research degree at the University of Bristol. First to my supervisor, Professor Martin Genner, for his tremendous help and support throughout my project, especially in the transition to remote working under unprecedented circumstances. I would also like to thank fellow lab colleagues who got involved and supported me in this project, especially Dr Rupert Collins who aided in my laboratory training and morphological identification of specimens. Thanks also to Dr Chris Clements for his involvement in my six-month assessment and constructive review in the midpoint of my project. Finally, I would like to acknowledge and thank all those who supported me through my academic development, specifically my family and the friends whom I met during my time in Bristol. iii Contents Student Declaration………………………………………………………………………...…..i Thesis Abstract………………………………………………………………………………...ii Acknowledgements…………………………………………………………………………...iii Table of Contents……………………………………………………………………………..iv List of Tables………………………………..……………………………….…...………….vii List of Figures………………………………..…………………….…………...…………...viii List of Appendices……………………………………………………………………………ix Chapter One 1.1 African freshwater biodiversity……………………………………………………………1 1.2 Threats to African freshwater biodiversity 1.2.1 Eutrophication……………………...........………..……………………………..3 1.2.2 Climate change……………………………..………………...……….….….…..4 1.2.3 Invasive species………………………………………………………………….5 1.3 Freshwater ichthyofauna of Tanzania……………………………………………………..5 1.4 Catfish biodiversity…………………………………..…….………….....………..….…...6 iv 1.5 Morphological diversity of African freshwater fishes 1.5.1 Sympatry………………………………………………………………………...7 1.5.2 Allopatry…………………………………………………………………………8 1.5.3 Cryptic species…………………………………………………………………...9 1.6 DNA Barcoding and molecular diversity 1.6.1 Background…………………………………….……….…....………..……….10 1.6.2 Barcoding African freshwater fishes…………………………………………...11 1.6.3 Choice of DNA barcode………………………………………………………..12 1.7 Morphological vs molecular data in fish phylogenetics………………………………….13 Chapter Two 2.1 Abstract………………….…………..…………………………………………………...15 2.2 Introduction…….…..………………...………..….…………………………..….………16 2.3 Materials and methods……………………...………..….…………………..…………...18 2.4 Results………………………..……………...………..….………………………………23 2.5 Discussion………………………………………………………………………………..27 Chapter Three 3.1 Abstract………………….…………..…………………………………………………...33 v 3.2 Introduction…….…..………………...………..….…………………………..….………34 3.3 Materials and methods……………………...………..….…………………..…………...38 3.4 Results………………………..……………...………..….………………………………40 3.5 Discussion………………………………………………………………………………..47 Chapter Four 4.1 DNA barcoding for resolution of East African freshwater fish biodiversity…………….51 4.2 The future of Chiloglanis study…………………………………………………………..52 4.3 Concluding remarks……………………………………………………………………...55 References……………………………………………………………………………………56 vi List of Tables Table 2.1. Collection information for specimens…………………………………………….20 Table 2.2. DNA barcode genetic distances…………………………………………………..24 Table 3.1. Summary details of morphological samples……………………………………...37 Table 3.2. Genetic clades recovered by site………………………………………………….39 Table 3.3. Morphometric measurements for Chiloglanis specimens………………………...41 Table 3.4. Logged residual measurements from sympatric clades…………………………...44 Table 3.5. Results of Anova (Type II)………………………………………………………..47 vii List of Figures Figure 2.1. Map detailing the construction site of the Stiegler’s Gorge……………………..17 Figure 2.2. COI neighbour-joining tree phylogeny…………………………………………..25 Figure 2.3. 12S neighbour-joining tree phylogeny…………………………………………..26 Figure 2.4. Histograms of genetic distance for DNA barcodes………………………………27 Figure 3.1. Maximum likelihood alongside representative specimens………………………36 Figure 3.2. Principal Component Analysis of all specimens………………………..……….40 Figure 3.3. Principal Component Analyses of sympatric clades……………………………..43 Figure 3.4. Principal Component Analyses of allopatric populations………………………..45 Figure 3.5. Principal Component Analyses of allopatric populations with overlaps………...46 Figure 4.1. Diagram of a generalized Amphilius specimen…………………………………..54 viii List of Appendices Appendix 2.1. DNA barcoding database search results……………………………………...67 Appendix 3.1. Collection sites for Chiloglanis specimens…………………………………..70 Appendix 3.2. Illustration depicting the linear morphometric measurements……………….71 ix CHAPTER ONE: GENERAL INTRODUCTION. 1.1 African freshwater biodiversity It is estimated that around 42% of the world’s 32,500 fish species live in freshwater habitats, despite these ecosystems occupying only 0.8% of the Earth’s surface (Darwall et al., 2005; Nelson, 2006). Africa has at least 3,300 freshwater species, representing one of the most unique and diverse ichthyofaunas globally. Its species richness is only slightly less than that of Asia (~3,600) and South America (~4,200) (Nyboer et al., 2019). Complex geological processes including uplift, volcanic activity, the formation of deserts and tectonic rifting led to substantial biogeographical