Morphological and molecular approaches to the management of captive antelopes By Cristiane Bastos Silveira A thesis submitted for fulfilment of the Degree of Doctor of Philosophy University of London, 2002 ProQuest Number: U643625 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. uest. ProQuest U643625 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 To my daughters Flor and Concha I declare that this thesis represents my own original work, and that no part of this thesis has been, or is being, submitted for any other diploma, or degree, qualification. Cristiane Bastos Silveira IV Abstract The aims of the present study were to generate morphological and genetic data to support management strategies for captive antelope populations. To achieve this objective I i) tested the validity of previously described subspecies using morphological characters; ii) tested the utility of the external morphological characters to provenance specimens in captive populations; iii) developed genetic markers for characterising subspecies diversity within collections; iv) identified and characterised the genetic diversity in the captive populations compared to wild specimens; and v) tested the power of nuclear genetic markers to uncover relationships within groups. Morphometric data, based on nineteen skull and horn characters, were used for multivariate analyses of Aepyceros melampus (impala) populations. A quantitative method for skin pattern analysis based on standard colour charts was developed. The results supported four out of five described subspecies: A. m. melampus, A. m. petersi, A. m. johnstoni, and A. m. suara. Mitochondrial DNA sequences proved to be a powerful marker to assess the subspecies diversity within collections, and data revealed hybrid stock of A. m. petersi x A. m. melampus in European Zoos. A. m. petersi is listed as vulnerable and its status in captivity gives cause for great concern and the need for urgent measures. Museum specimens proved to be a reliable source of historical genetic information, giving DNA sequences on wild impala populations back to 70 years ago. Ten Bos taurus (cattle) micro satellite loci were successfully amplified in five study species: A. melampus (impala); Hippotragus niger (sable antelope); H. equinus (roan antelope); Kobus ellipsiprymnus (waterbuck) and K. leche (leche). In six loci the allele size range was diagnostic at the species and/or genus level. These findings are especially important for the investigation of hybridisation, studies on mixed groups, and material of unknown provenance. Simulation of parentage inference, based on the 6 polymorphic loci, indicates that this set of markers is able to resolve parentage between two candidate parents, at 95% confidence, whether one parent is known (99% certainty) or not (98%). The present study was able to test the accuracy of these simulations using a captive group of 24 impalas. The results strongly support the use of this analysis to assess the number of markers or average polymorphism required to resolve parentage or relatedness in captive population studies. Acknowledgements I am greatly indebted to: My supervisors, Adrian Lister and Michael Bruford, for their unceasing support, encouragement, patience and valuable advice; Maria do Mar Oom for her unceasing support and friendship, providing laboratory facilities, and allowing the continuation of the genetic work in Lisbon University; All the European Zoos that supported my work by sending samples and maintaining an interest in the results. Special thanks to Lisbon Zoo and staff for continuing support; The staff at the Institute of Zoology, Zoological Society of London, who helped and supported me during my training with molecular genetic techniques, specially Dave Cheesman, Dada Gotelli and Michelle Bayes; My London colleagues Russell Seymour, Lounes Chikhi, Benoit Goossens and Stephan Funk, who made my working time more pleasant and always provided help when I most needed it; Paula Jenkins of the British Natural History Museum for making accessible the antelope specimens kept by the museum, and Richard Sabin for teaching me techniques of specimen sampling for DNA extraction; The friendly staff of the Powell Cotton Museum in Kent, who allowed me to work with their antelope specimens and made available all the samples for DNA extraction that I requested. I will always be indebted for such exceptional support that I, and other colleagues, have received from this Museum; My colleagues Helen, Kath, Mireille, Nick and Stephen from Cardiff University, who made my time in the lab warmer, specially Carlos for long hours of Portuguese conversation about football, life and jobs; Diodalia Dias for her friendship and support, and making available her laboratory at Lisbon University; Pedro Rodrigues, Cristina Luis, Octavio Paulo and Nick Anthony for their suggestions with the data analysis; VI Dona Branca for her friendship, football talking and helping me to find the large and small laboratory itens that I needed in Lisbon University. Special thanks to Lourdes and Eugenia for always helping me in the library; My friends Ana Rita, Cristina, Joana Morais, Joana Vacas and Patricia who made my life easier and funnier when I was running against time to accomplish my endless deadlines; My family in Brazil who even from a distance always gave me support and encouragement to achieve this big task; My cousin Ivoninha, wherever you are, this is an achievement that I share with you; And to Henrique, for never letting me down. You will always have my love and admiration. This work had the financial support of the Portuguese Foundation for Technology and Science through the grant PRAXIS D/13421/97. v u TABLE OF CONTENTS Page DECLARATION iii ABSTRACT iv ACKNOWLEDGEMENTS v TABLE OF CONTENTS vii LIST OF TABLES xii LIST OF FIGURES xvi CHAPTER ONE - Management of wild animais in captivity 1 1.1 Aims of the study 2 1.2 Captive populations 3 1.3 The small population paradigm 5 1.4 Defining units of management 6 1.5 Preserving genetic diversity 8 1.6 Tools for management of captive populations 9 1.6.1 The use of morphological data 10 1.6.2 The use of genetic data 11 CHAPTER TWO - Antelopes: their status in the wild and in captivity 14 2.1 Introduction 15 2.2 Antelope status in the wild 20 2.3 Antelopes in European zoos 23 CHAPTER THREE - Study taxa 29 3.1 Introduction 30 3.2 Gtrms Aepyceros 32 3.2.1 Aepyceros melampus Lichtenstein, 1812 32 3 3 Gqiwis Hippotragus 38 3.3.1 Hippotragus niger (\\2ims, 1838) 38 33.2 Hippotragus equinus 44 3.4 Genus Kobus 51 3.4.1 Kobus ellipsiprymnus {Pg\\hy,\^2>2>) 51 3.4.2 Kobus leche Gv?iy,\^5Q 57 Vlll CHAPTER Y0\5K-Aepyceros melampus geographical variation and subspecies 63 validation based on morphological characters 4.1 Introduction 64 4.2 Material and methods 66 4.2.1 Samples 66 4.2.2 Sexing and ageing of specimens 67 4.2.3 Skull measurements 68 4.2.4 Horn measurements 68 4.2.5 Coat patterns analysis 71 4.2.6 Statistical analysis 73 4.2.6.1 Descriptive statistics 74 4.2.6.2 Univariate analysis 74 4.2.6.3 Multivariate analysis 75 4.3 Results 77 4.3.1 Age and sex variation 77 4.3.2 Taxonomic and geographical variation 89 4.3.2.1 Male skull and horn characters 89 • Exploring PCA clusters 94 • Testing variation among geographic groups 97 • Testing variation among subspecies 108 4.3.2.2 Female skull characters 113 4.3.2.3 Coat patterns 117 4.4 Discussion 124 CHAPTER FIVE - Phylogeographic matching of captive antelopes based on 129 mitochondrial DNA sequences: Impala case study. 5.1 Introduction 130 5.2 Mitochondrial DNA 131 5.2.1 Control region 132 5.3 Mitochondrial DNA studies on African antelopes 133 5.4 Impalas in European Zoos 137 5.5 Aims of the study 138 5.6 Material and methods 139 5.6.1 Samples 139 5.6.1.1 Captive populations 139 IX 5.6.1.2 Museum specimens 139 5.6.2 DNA extraction 140 5.6.2.1 Whole blood and tissue 140 5.6 2.2 Museum material 140 5.6.3 DNA Amplification 141 5.6.4 Sequencing 142 5.6.5 Sequencing from GenBank 142 5.6.6 Data analysis 143 Results 145 5.7.1 Haplotype and nucleotide diversity 145 5.7.2 Genetic distance 149 5.7.3 Phylogeographic matching 150 5.73.1 Neighbour-joining 150 S.7.3.2 Parsimony 153 Discussion 157 CHAPTER SIX - The use of bovine primers for analysing microsatellite 160 variation in five species of African antelopes. 6.1 Introduction 161 6.1.1 Cross-species amplification 162 6.1.2 Microsatellites applied to antelope species 164 6.2 Aims of the study 165 6.3 Material and methods 166 6.3.1 Samples 166 63.1.1 Captive populations 166 6.3.1.2 Museum/wild specimens 166 6.3.2 Microsatellite loci 167 6 3 3 DNA Extraction 167 6.33.1 Whole blood and tissue 167 633.2 Museum material 167 63.4 DNA amplification 167 6.3.5 Genotyping 170 6.3.6 Cloning and sequencing 170 63.7 Data analysis 171 6.4 Results 172 6.4.1 Amplification rate 172 6.4.2 Allelic diversity 174 6.4.3 Species allele size range 176 6.4.4 Sequences of alleles 182 6.5 Discussion 185 CHAPTER SEVEN - The use of microsatellites for paternity assignment in 190 captive populations: Impala case study.