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Genetic analysis of the critically endangered Trinidad Piping guan (Pipile pipile): Implications for phylogenetic placement and conservation strategies Item Type Thesis or dissertation Authors Robinson, Louise A. Publisher University of Chester Download date 27/09/2021 02:16:54 Link to Item http://hdl.handle.net/10034/346901 This work has been submitted to ChesterRep – the University of Chester’s online research repository http://chesterrep.openrepository.com Author(s): Louise Anne Robinson Title: Genetic analysis of the critically endangered Trinidad Piping guan (Pipile pipile): Implications for phylogenetic placement and conservation strategies Date: November 2011 Originally published as: University of Chester PhD thesis Example citation: Robinson, L. A. (2011). Genetic analysis of the critically endangered Trinidad Piping guan (Pipile pipile): Implications for phylogenetic placement and conservation strategies. (Unpublished doctoral dissertation). University of Chester, United Kingdom. Version of item: Amended version Available at: http://hdl.handle.net/10034/346901 Genetic analysis of the critically endangered Trinidad Piping guan (Pipile pipile ): Implications for phylogenetic placement and conservation strategies Thesis submitted in accordance with the requirements of the University of Chester for the degree of Doctor of Philosophy By: Louise Anne Robinson November 2011 Acknowledgements This thesis is dedicated to my husband Anthony for his constant support throughout the course of this research. I would like to thank the University Gladstone Fellowship scheme for funding this PhD research from January 2008 to January 2011 and the World Pheasant Association for their continued support and Research Associate status awarded to me in 2008. Sincere thanks are owed to my principle supervisor Dr Ian McDowall whose unfailing academic and personal support has been essential to the successful completion of this project. I also thank my joint supervisor Dr Charlotte Hosie for her commitment to the research and providing valuable feedback throughout the course of study. This research would not have been possible without the help of Prof. John Cooper and the support of the Pawi Study Group in Trinidad who provided genetic samples of the Trinidad Piping guan and information regarding current issues. Photographs of the Trinidad Piping guan and related pictures supplied by Margaret Cooper and Kerrie T. Naranjit are highly appreciated for there visual demonstrations of issues discussed in the text. Dr Luís Fábio Silveira and Patrícia Lopes at the Universidade de São Paulo are thanked for their provision of samples from Cracid species. Special thanks are given to Patrícia Lopes for her advice on the morphology of Piping guans. Also acknowledged for their donation of samples is Chester Zoological Society who provided feathers from the red-billed curassow (Crax blumenbachii ) and black vulture (Coragyps atratus ) for use as comparator species. For the investigation of closely related species, the Lotherton Bird Garden, Cotswold Wildlife Park and Gardens and Fenton Barns are acknowledged for the provision of feather samples against which methods devised in the current research were tested. I would also like to thank my family for their boundless support and PhD students past and present for continual encouragement. Special appreciation is given to two friends in particular. Robert Swain is thanked for his friendship, support and assistance with computer software without which the progress of this research would have been significantly impeded. Additionally, Robert Coleman with his infectious positive attitude and generosity is thanked for creating an excellent working environment. Finally, I would like to honour the memory of my grandparents and of family friend Harry Shaw. I also thank Harry’s partner June Bayley for providing chicken feathers on which many of the universal primers were originally tested. Table of Contents Page N o: Abbreviations Used in the Text .................................................................... 1 List of Tables and Figures............................................................................. 3 Abstract ......................................................................................................... 15 Chapter 1 Conservation and Status of the Trinidad Piping guan 1.1 Cracids..................................................................................................... 16 1.1.1 The Piping guans ........................................................................ 17 1.1.2 Disputed Classification of the Piping guans ................................ 18 1.1.3 Current phylogeny of the Piping guans ....................................... 19 1.1.4 Geographical distribution of the Trinidad Piping guan................. 21 1.1.5 Morphology of the Trinidad Piping guan...................................... 23 1.1.6 History and ecology of the Trinidad Piping guan ......................... 24 1.2 Conservation of the species .................................................................. 25 1.2.1 Raising public awareness............................................................ 25 1.2.2 The effect of awareness campaigns on attitudes ........................ 27 1.2.3 Ecotourism .................................................................................. 28 1.2.4 Field based research................................................................... 31 1.2.5 The use of species field markers in Trinidad ............................... 32 1.2.6 Combating wildlife crime ............................................................. 33 1.3 Forensic Techniques .............................................................................. 34 1.3.1 Mitochondrial DNA ...................................................................... 35 1.3.2 Evidence of small populations within mitochondrial DNA ............ 38 1.4 Aims of the investigation ....................................................................... 39 Page N o: Chapter 2 General Methodologies and Materials 2.1 Introduction to general procedures ...................................................... 41 2.2 Biological samples used for DNA extraction........................................ 41 2.2.1 Further taxa and permits obtained during the course of study .... 42 2.3 DNA extraction ........................................................................................ 44 2.4 Mitochondrial genome regions sequenced in the current study........ 45 2.4.1 DNA amplification........................................................................ 45 2.4.2 ND2 Universal Primers................................................................ 45 2.4.3 Degenerate bases....................................................................... 46 2.4.4 Cytochrome b Universal primers ................................................. 46 2.4.5 Principles of amplification/sequencing primer design.................. 47 2.4.6 Primers used during the course of the study ............................... 48 2.5 Electrophoresis....................................................................................... 51 2.3 Cycle sequencing and data handling.................................................... 52 Chapter 3 Review and establishment of sampling methodologies for use in the current investigation 3.1 Analysis of sampling methodologies for the species of study........... 53 3.2 Museum sampling...................................................................................54 3.2.1 Maintaining museum collections.................................................. 54 3.2.2 Extraction of DNA from museum samples................................... 55 3.2.3 Trinidad Piping guan museum samples ...................................... 56 Page N o: 3.3 The importance of sampling methodology selection in endangered species research........................................................... 57 3.3.1 Invasive sampling........................................................................ 60 3.3.2 Non-invasive sampling ................................................................ 61 Methods 3.4 Comparison of blood and feather sampling......................................... 62 3.4.1 The viability of invasive sampling to the current study................. 64 3.5 Feather samples as a suitable DNA resource ...................................... 64 3.5.1 Non-invasive feather sample collection....................................... 65 3.5.2 Examination of feathers prior to genetic testing .......................... 66 3.5.3 Common feather types in genetic studies ................................... 67 3.5.4 Location of highest DNA yield within a feather ............................ 68 3.5.5 The integrity of non-invasive feather samples ............................. 70 3.5.6 Increasing the chance of DNA extraction in non-invasive Sampling ..............................................................................................71 3.6 DNA extraction, amplification and subsequent electrophoresis ........ 72 3.7 Methodology selection for the current investigation........................... 72 3.7.1 Results of the chosen sampling methodology ............................. 73 3.8 Discussion............................................................................................... 79 3.9 Conclusion .............................................................................................. 81 Page N o: Chapter 4 Comparative sequence analysis and phylogenetic