GENETIC VARIATION OF THE FOREST ELEPHANT Loxodonta africana cyclotis ACROSS

244 Pages·2013·14.1 MB·English

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.
S ig n e d c ^ ^ ^ p f r ^ f r f t...................(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.
...A.2 ^ . . 0 . 5 .
Sqm e d r . (Candidate) Date
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^^—
\ \
X ! V.
v A •7^>- • \ A L .0.. I
Signed ... .T. .7. . .\. . (Candidate) Date
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. ^
.. . L.. . .D. A.. 0. .
Signed , .. ... (Candidate) Date :
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................................................................ 46
vi
2.0 ABSTRACT............................................................................................................. 46
2.1 STUDY SITES........................................................................................................ 46
2.2 SAMPLING STRATEGY..................................................................................... 48
2.2.1 Sample collection and training of field researchers............................. 51
2.2.2 Non-invasive sampling............................................................................. 51
2.2.3 Dung sample preservation......................................................................... 53
2.2.4 Extraction..................................................................................................... 53
Faecal extraction....................................................................................... 54
Blood and skin tissue extraction............................................................. 54
2.3 MOLECULAR TECHNIQUES........................................................................... 55
2.3.1 Mitochondrial DNA (D-Loop and cytochrome b) sequencing 55
Amplification.............................................................................................. 56
Sequencing.................................................................................................. 56
Cloning........................................................................................................ 57
2.3.2 Microsatellite analysis.............................................................................. 57
Screening..................................................................................................... 57
Multiplex constitution............................................................................... 59
Genotyping criteria................................................................................... 60
2.4 DATA ANALYSIS................................................................................................ 62
2.4.1 Mitochondrial.............................................................................................. 62
Sequence alignment.................................................................................. 62
Genetic diversity........................................................................................ 62
Genetic differentiation and analysis of molecular variance............... 62
Phylogenetic relationships....................................................................... 63
Analysis of population demography....................................................... 64
2.4.2 Nuclear microsatellite loci........................................................................ 64
Genetic diversity analysis......................................................................... 65
Analysis of molecular variance................................................................ 66
Population structure and genetic admixture analysis......................... 66
2.5 REFERENCES....................................................................................................... 68
CHAPTER 3 POPULATION GENETIC STRUCTURE OF FOREST
ELEPHANTS IN WEST CENTRAL AFRICA BASED ON
MITOCHONDRIAL DNA CONTROL REGION VARIATION 74
3.0 ABSTRACT............................................................................................................ 74
3.1 INTRODUCTION................................................................................................. 74
vii
3.2 MATERIALS AND METHODS......................................................................... 78
3.2.1 Samples...................................................................................................... 78
3.2.2 Laboratory procedures............................................................................ 82
3.2.3 Analysis of genetic diversity and differentiation................................ 82
3.2.4 Analysis of population demography..................................................... 83
3.3 RESULTS................................................................................................................ 83
3.3.1 Sequences and haplotypes analysis....................................................... 83
3.3.2 Genetic diversity and population structure.......................................... 92
3.3.3 Population demography.......................................................................... 96
3.4 DISCUSSION......................................................................................................... 104
3.5 REFERENCES....................................................................................................... 110
CHAPTER 4 COMPLEX PHYLOGEOGRAPHIC HISTORY OF CENTRAL
AFRICAN FOREST ELEPHANT AND ITS IMPLICATIONS
FOR TAXONOMY........................................................................................ 117
4.0 ABSTRACT............................................................................................................. 117
4.1 INTRODUCTION.................................................................................................. 118
4.2 MATERIALS AND METHODS......................................................................... 120
4.2.1 Sampling and laboratory procedures.................................................... 120
4.2.2 Analysis of genetic diversity and differentiation................................ 122
4.2.3 Analysis of population demography..................................................... 122
4.3 RESULTS................................................................................................................ 123
4.3.1 Central forest samples............................................................................. 123
4.3.2 Genetic diversity...................................................................................... 123
4.3.3 Population structure................................................................................. 125
4.3.4 Demographic history................................................................................ 133
4.4 DISCUSSION......................................................................................................... 136
4.5 REFERENCES....................................................................................................... 139
CHAPTER 5 POPULATION GENETIC STRUCTURE OF FOREST
ELEPHANTS IN WEST CENTRAL AFRICA BASED ON
MICROSATELLITE LOCI........................................................................ 141
5.0 ABSTRACT........................................................................................................... 141
5.1 INTRODUCTION.................................................................................................. 142
5.2 MATERIALS AND METHODS......................................................................... 145
5.2.1 Study area................................................................................................... 145
5.2.2 Sampling and DNA amplification......................................................... 147
5.2.3 Population genetic analyses.................................................................... 148
viii
5.3 RESULTS................................................................................................................ 150
5.3.1 Genetic diversity........................................................................................ 150
5.3.2 Deviation from Hardy-Weinberg Equilibrium and LD....................... 151
5.3.3 Population structure.................................................................................. 152
5.4 DISCUSSION......................................................................................................... 161
5.5 REFERENCES....................................................................................................... 166
CHAPTER 6 GENERAL DISCUSSION........................................................................... 174
6.1 TWO-SPECIES MODEL VERSUS A COMPLEX EVOLUTIONARY
HISTORY OF AFRICAN ELEPHANT............................................................. 177
6.2 CONSERVATION IMPLICATIONS FOR CENTRAL AFRICAN
FOREST ELEPHANTS........................................................................................ 180
6.3 REFERENCES....................................................................................................... 183
APPENDIX................................................................................................................................ 186
APPENDIX 1......................................................................................................... 187
APPENDIX 2......................................................................................................... 188
APPENDIX 3......................................................................................................... 191
APPENDIX 4......................................................................................................... 197
APPENDIX 5......................................................................................................... 201
APPENDIX 6......................................................................................................... 206
APPENDIX 7......................................................................................................... 212
APPENDIX 8......................................................................................................... 215
ix