GENETIC VARIATION OF THE FOREST ELEPHANT Loxodonta africana cyclotis ACROSS CENTRAL AFRICA MIREILLE BA WE JOHNSON A thesis submitted to Cardiff University for the higher degree of Doctor of Philosophy June 2008 Cardiff School of Biosciences Cardiff University UMI Number: U585163 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U585163 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 I dedicate this thesis to my lovely children, Line and Franck-Olivier, my sisters: Louise, Irene, Isabelle and my late sister Yvonne; to my brothers: Freddy, Richard, William, Christian, Hughes and Gilles; to my mother Georgeline and my father John for their love, support and encouragement; finally to all my nieces and nephews. Akewa m 'polo! DECLARATION This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. c. Signedc^^^pfr^frft ................... (Candidate) Date .. \X >.. .'.Ai\. STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of PhD. Signed T.. \ . \ . .. V T ^ T . 7T77. (Candidate) Date ... \ . X..'.. \ ......Q 5) STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other spurces are acknowledged by explicit references. Sqm e d r . (Candidate) Date ...A.2^ ..0 .5 . STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter- library loan, and for the title and summary to be made available to outside organisations^^— \ \ ! V . Signed ... .T. .7. v A. .\. X. •7^>- • (Candidate) Date \ AL .0.. I STATEMENT 4: PREVIOUSLY APPROVED BARON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access previously approved by the Graduate Developmen^Committee. ^ Signed , .. ... (Candidate) Date .. L..:. .D. A.. 0. ACKNOWLEDGMENTS I am most grateful to Professor Michael W. Bruford, Dr. Jean E. Wickings and Dr. Benoit Goossens whose guidance, comments, commitment and encouragement made this work a success. Special thanks to my friend Stephen Clifford who helped me in many ways to successfully complete this project. I am grateful to Nicole Muloko and Nicola Anthony who gave me intensive training in molecular biology techniques. Special gratitude also goes to Joanna Cable and Joanna Setchell who kindly accepted to correct my grammar. To all my current and former lab mates and friends: Muya, Gaby, Rhys, Farina, Pati, Milena, Renata, Coralie, Geoff, Tim, Jeff, Viola, Andy, Ben, David, Dan, Kath, Angela, Fairus, Yoshan, Erica, Simon, Trini, Carlos, Elis and Sofia. My interactions and discussions with you all was always a joy, which nourished my motivation. Special thanks also to UGENET-CIRMF: Marie, Michel, Christiane, Simon, Paul, Celine, and all people at CIRMF especially my friends Odile and Maria for dways giving me their support. Finally, to my best friends Edith and Laure. This project was supported by grants from US Fish and Wildlife Service, French cooperation/FORINFO, Darwin Initiative scholarship, British Ecological Society, Tusk Trust and Centre International de Recherches Medicales in Franceville. IV SUMMARY The first comprehensive genetic study of central African forest elephants (CAEs) is presented here based on mitochondrial DNA (mtDNA) and nuclear microsatellite loci. MtDNA analysis revealed low genetic divergence between most groups. Haplotype distribution was not correlated with geographical localities, indicating high levels of gene flow. Two divergent haplogroups, illustrated by a bimodal distribution of pairwise differences in the control region, implies that secondary contact and ongoing introgression has occurred between populations expanding from at least two putative glacial refugia. Similarly, microsatellite analysis revealed low genetic differentiation among sites, suggesting high levels of gene flow as well as regional admixture with two genetically-based clusters inferred from Bayesian analyses. It is important to note through, that although both mtDNA and microsatellites identified two groups or genetic clusters, assignment of hdividuals to these clusters was not consistent across genomes possibly a result of differential admixture in nuclear and mitochondrial DNA No correlation was found between genetic and geographical distances for both genomes. Previous phylogenetic analyses, using either on genetic or morphological characters, were based on a very limited number of forest elephant samples. A large-scale re­ assessment of mitochondrial DNA diversity in CAEs compared to published data on both forest and savannah forms revealed a complex phylogeographic history for African elephants, and an evolutionary trajectory more complex than prevailing two-taxon models have assumed. Mitochondrial control region and Cytochrome b sequences were analysed for CAEs and compared to other African elephant data. CAE populations fell into at least two lineages with West African elephants (both forest and savannah) sharing their mitochondrial history almost exclusively with Central African forest elephants. Extant African elephant populations therefore seem to have originated from multiple refugia lineages that have subsequently undergone introgression. Thus, the complex phylogeographic history of African elephants does not support a simple two-taxon model and management strategies incorporating the two-taxa model could be misinformed until further data give clarifies the origins of elephant populations throughout Africa. TABLE OF CONTENTS DECLARATION...................................................................................................................... iii ACKNOWLEDGEMENTS................................................................................................... iv SUMMARY................................................................................................................................ v CONTENTS............................................................................................................................... vi LIST OF TABLES................................................................................................................... x LIST OF FIGURES................................................................................................................. xii CHAPTER! GENERAL INTRODUCTION.................................................................. ! 1.1 EVOLUTION OF PROBOSCIDEA.................................................................... 1 1.2 THE TAXONOMY OF MODERN ELEPHANT: LOXODONTA............... 5 1.3 MORPHOLOGICAL DIFFERENCES.............................................................. 7 1.3.1 African elephant versus Asian elephant ................................................ 7 1.3.2 African forest elephants versus African savannah elephants ............ 8 1.3.3 West African elephants......................................................................... 9 1.3.4 The distribution of Loxodonta ................................................................ 10 1.4 THE CENTRAL AFRICAN FOREST ELEPHANT: L. AFRICANA CYCLOTIS (MATSCHIE, 1900)........................................................................ 12 1.4.1 Distribution ................................................................................................ 13 1.4.2 Ecology-Habitat / Diet ............................................................................. 14 1.4.3 Reproduction .............................................................................................. 15 1.4.4 Social structure .......................................................................................... 16 1.4.5 Movements ................................................................................................. 18 1.4.6 Poaching and habitat loss ........................................................................ 19 1.5 JUSTIFICATION OF THE STUDY................................................................... 20 1.6 GENETICS.............................................................................................................. 22 1.6.1 Choice of molecular marker .................................................................... 22 Polymerase Chain Reaction using non-invasive samples ................... 22 Mitochondrial DNA (mtDNA) .................................................................. 23 Nuclear integrations of mitochondrial DNA (Numts) .......................... 24 Microsatellite DNA ................................................................................... 25 1.7 AN OVERVIEW ON AFRICAN ELEPHANT GENETIC STUDIES 25 1.8 HYPOTHESES AND AIMS ................................................................................ 28 1.9 REFERENCES....................................................................................................... 31 CHAPTER 2 MATERIALS AND METHODS...............................................................